Use of vapor deposition coated flow paths for improved chromatography of metal interacting analytes

ABSTRACT

R1, R2, R3, R4, R5, and R6 are each independently selected from (C1-C6)alkoxy, —NH(C1-C6)alkyl, —N((C1-C6)alkyl)2, OH, ORA, and halo. RA represents a point of attachment to the interior surfaces of the fluidic system. At least one of R1, R2, R3, R4, R5, and R6 is ORA. X is (C1-C20)alkyl, —O[(CH2)2O]1-20—, —(C1-C10)[NH(CO)NH(C1-C10)]1-20-, or —(C1-C10)[alkylphenyl(C1-C10)alkyl]1-20-.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of U.S. provisionalapplication No. 62/559,895 filed Sep. 18, 2017 entitled “Use of VaporDeposition Coated Flow Paths for Improved Chromatography ofBiomolecules,” the contents of which is incorporated herein by referencein its entirety.

FIELD OF THE TECHNOLOGY

This technology relates to the use of vapor deposition coated flow pathsfor improved chromatography. More specifically, this technology relatesto chromatographic devices for separating analytes in a sample havingcoated flow paths, methods of separating analytes in a sample (forexample, glycans, peptides, pesticides, and citric acid cyclemetabolites) using a fluidic system that includes coated flow paths, andmethods of tailoring a fluidic flow path for separation of a sample.

BACKGROUND OF THE TECHNOLOGY

Analytes that interact with metal have often proven to be verychallenging to separate. The desire to have high pressure capablechromatographic systems with minimal dispersion has required that flowpaths decrease in diameter and be able to withstand increasingly highpressures at increasingly fast flow rates. As a result, the material ofchoice for chromatographic flow paths is often metallic in nature. Thisis despite the fact that characteristics of certain analytes, forexample, biomolecules, proteins, glycans, peptides, oligonucleotides,pesticides, bisphosphonic acids, anionic metabolites, and zwitterionslike amino acids and neurotransmitters, are known to have unfavorableinteractions, so called chromatographic secondary interactions, withmetallic surfaces.

The proposed mechanism for metal specific binding interactions requiresan understanding of the Lewis theory of acid-base chemistry. Pure metalsand metal alloys (along with their corresponding oxide layers) haveterminal metal atoms that have characteristics of a Lewis acid. Moresimply, these metal atoms show a propensity to accept donor electrons.This propensity is even more pronounced with any surface metal ionsbearing a positive charge. Analytes with sufficient Lewis basecharacteristics (any substance that can donate non-bonding electrons)can potentially adsorb to these sites and thus form problematicnon-covalent complexes. It is these substances that are defined asmetal-interacting analytes.

For example, analytes having phosphate groups are excellent polydentateligands capable of high affinity metal chelation. This interactioncauses phosphorylated species to bind to the flow path metals thusreducing the detected amounts of such species, a particularlytroublesome effect given that phosphorylated species are frequently themost important analytes of an assay.

Other characteristics of analytes can likewise pose problems. Forexample, carboxylate groups also have the ability to chelate to metals,albeit with lower affinities than phosphate groups. Yet, carboxylatefunctional groups are ubiquitous in, for example, biomolecules, givingthe opportunity for cumulative polydentate-based adsorptive losses.These complications can exist not only on peptides and proteins, butalso glycans. For example, N-glycan species can at times contain one ormore phosphate groups as well as one or more carboxylate containingsialic acid residues. Additionally, smaller biomolecules such asnucleotides and saccharides, like sugar phosphates, can exhibit similarbehavior to the previously mentioned N-glycan molecules. Moreover,chromatographic secondary interactions can be especially problematicwith biomolecules, particularly larger structures, because they have acapacity (via their size and structural order) to form microenvironmentsthat can adversely interact with separation components and flow pathsurfaces. In this case, a biomolecule or analyte having largerstructures, can present structural regions with chemical properties thatamplify a secondary interaction to the material of a flow path. This,combined with the cumulative metal chelation effects curtails theoverall effective separation of biomolecules, pesticides, bisphosphonicacids, anionic metabolites, and zwitterions like amino acids andneurotransmitters.

An alternative to using metal flow paths is to use flow pathsconstructed from polymeric materials, such as polyether ether ketone(PEEK). PEEK tubing, like most polymeric materials, is formed by meansof an extrusion process. With polymeric resin, this manufacturingprocess can lead to highly variable internal diameters. Accordingly,PEEK column hardware yields unfavorable differences in the retentiontimes as can be observed from switching between one column and the next.Often, this variation can be a factor of three higher than a metalconstructed column. In addition, the techniques for fabricating polymerbased frits are not yet sufficiently optimized to afford suitably ruggedcomponents for commercial HPLC columns. For example, commerciallyavailable PEEK frits tend to exhibit unacceptably low permeability.

Ongoing efforts to reduce chelation and secondary chromatographicinteractions of analytes with metal chromatographic surfaces in aneffort to facilitate chromatographic separation having higherresolutions are therefore needed.

SUMMARY OF THE TECHNOLOGY

One advantage of the alkylsilyl coatings of the present technology isthat metal chromatographic flow paths can be used while minimizing theinteractions between analytes and the metal flow paths. Coating the flowpath of instrumentation and chromatographic devices with certainalklysilyl compositions improves multiple aspects of liquidchromatography separations where the analyte of interest is ametal-interacting analyte. The use of alkylsilyl coatings on metal flowpaths allow the use of metal chromatographic flow paths, which are ableto withstand high pressures at fast flow rates, while minimizing thesecondary chromatographic interactions between the analyte and themetal. Therefore, high pressure components can be manufactured out ofstainless steel or other metallic or high pressure material. Thesecomponents made of high pressure material can then be tailored in thatthe internal flow paths can be modified with a coating to address thehydrophobicity of the flow path and reduce secondary chromatographicinteractions.

Provided herein, therefore, are methods for isolating analytescomprising the use of vapor depositing one or more alklysilylderivatives to at least one component of a fluidic system to form abioinert or low-bind coating, and eluting the analyte through thesystem. Unlike ambient, liquid phase silanization, coatings which arevapor deposited tend to produce, more resilient modifications ofsubstrates with precisely controlled thicknesses. Also, because vapordeposition is a non-line-of-sight process, this leads to a more uniformcoating over substrate contours and complex surfaces. This advantageallows for coatings to be applied to flow paths with narrow internaldiameters and curved surfaces, therefore addressing the need forincreasingly high pressures at increasingly fast flow rates.

Also provided herein are methods of tailoring a fluidic flow path forseparation of a sample comprising an analyte that includes infiltratinga vaporized source of one or more alkylsilyl derivatives through thefluidic flow path to form a bioinert (or low-bind) coating andcontrolling temperature and pressure to deposit a first coating onwetted surfaces of the flow path.

Also provided are methods of tailoring a fluidic flow path forseparation of a sample including an analyte comprising assessing thepolarity of the analyte, selecting an appropriate alkylsilyl derivative,and adjusting the hydrophobicity of wetted surfaces of the flow path byvapor depositing the appropriate alkylsilyl derivative to form abioinert, low-bind coating.

Further provided herein are methods of improving baseline returns in achromatographic system comprising introducing a sample including ananalyte into a fluidic system comprising at least one vapor depositedalkylsilyl derivative to form a bioinert, low-bind coating, and elutingthe sample through the system.

The disclosed methods can be applied to stainless steel or othermetallic flow path components and provides a manufacturing advantageover alternative non-metallic or non-metallic lined components.

In one aspect, the technology includes a chromatographic device forseparating analytes in a sample. The device includes a sample injectorhaving a sample injection needle for injecting the sample into themobile phase, a sample reservoir container in fluid communication withthe sample injector, a chromatography column downstream of the sampleinjector, wherein the chromatography column having fluid connectors, andfluid conduits connecting the sample injector and the chromatographycolumn. Interior surfaces of the fluid conduits, sample injector, samplereservoir container, and chromatography column form a fluidic flow pathhaving wetted surfaces. At least a portion of the wetted surfaces of thefluidic flow path are coated with a alkylsilyl coating, wherein thealkylsilyl coating is inert to at least one of the analytes in thesample. The alkylsilyl coating has the Formula I:

R¹, R², R³, R⁴, R⁵, and R⁶ are each independently selected from(C₁-C₆)alkoxy, —NH(C₁-C₆)alkyl, —N((C₁-C₆)alkyl)₂, OH, OR^(A), and halo.R^(A) represents a point of attachment to the interior surfaces of thefluidic system. At least one of R¹, R², R³, R⁴, R⁵, and R⁶ is OR^(A). Xis (C₁-C₂₀)alkyl, —O[(CH₂)₂O]₁₋₂₀—, —(C₁-C₁₀)[NH(CO)NH(C₁-C₁₀)]₁₋₂₀-, or—(C₁-C₁₀)[alkylphenyl(C₁-C₁₀)alkyl]₁₋₂₀-. The device can include one ormore of the following embodiments in any combination thereof.

The alkylsilyl coating can have a contact angle of at least 15°. In someembodiments, the alkylsilyl coating has a contact angle less than orequal to 30° or less than or equal to 90°.

The chromatographic device can also include a detector downstream of thechromatography column. The fluidic flow path can also include thedetector. In some embodiments the detector is a mass spectrometer andthe fluidic flow path includes wetted surfaces of an electrosprayneedle.

In some embodiments, the fluidic flow path has a length to diameterratio of at least 20. The alkylsilyl coating can have a thickness of atleast 100 Å.

In some embodiments, X is (C₂-C₁₀)alkyl. In other embodiments, X isethyl. R¹, R², R³, R⁴, R⁵, and R⁶ can each be methoxy or chloro. Thealkylsilyl coating of Formula I can be bis(trichlorosilyl)ethane orbis(trimethoxysilyl)ethane.

In some embodiments, the device also includes a second alkylsilylcoating in direct contact with the alkylsilyl coating of Formula I. Thesecond alkylsilyl coating has the Formula II:

R⁷, R⁸, and R⁹ are each independently selected from —NH(C₁-C₆)alkyl,—N[(C₁-C₆)alkyl]₂, (C₁-C₆)alkoxy, (C₁-C₆)alkyl, (C₁-C₆)alkenyl, OH, andhalo. R¹⁰ is selected from (C₁-C₆)alkyl, —OR^(B),—[O(C₁-C₃)alkyl]₁₋₁₀O(C₁-C₆)alkyl, —[O(C₁-C₃)alkyl]₁₋₁₀OH, and phenyl,wherein said (C₁-C₆)alkyl is optionally substituted with one or morehalo and wherein said phenyl is optionally substituted with one or moregroups selected from (C₁-C₃)alkyl, hydroxyl, fluorine, chlorine,bromine, cyano, —C(O)NH₂, and carboxyl. R^(B) is —(C₁-C₃)alkyloxirane,—(C₁-C₃)alkyl-3,4-epoxycyclohexyl, or —(C₁-C₄)alkylOH. The hashed bondto R¹⁰ represents an optional additional covalent bond between R¹⁰ andthe carbon bridging the silyl group to form an alkene, provided y is not0. y is an integer from 0 to 20.

In some embodiments, y is an integer from 2 to 9. In some embodiments,is 9, R¹⁰ is methyl, and R⁷, R⁸, and R⁹ are each ethoxy or chloro.

The alkylsilyl coating of Formula II can be(3-glycidyloxypropyl)trimethoxysilane, n-decyltrichlorosilane,trimethylchlorosilane, trimethyldimethyaminosilane,methoxy-polyethyleneoxy(1-10) propyl trichlorosilane, ormethoxy-polyethyleneoxy(1-10) propyl trimethoxysilane. In someembodiments, the alkylsilyl coating of Formula II is(3-glycidyloxypropyl)trimethoxysilane followed by hydrolysis.

The alkylsilyl coating of Formula I and II can provide a desired contactangle of about 0° to about 105°.

In some embodiments, the alkylsilyl coating of Formula I isbis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane and thealkylsilyl coating of Formula II is(3-glycidyloxypropyl)trimethoxysilane. The alkylsilyl coating of FormulaI can be bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane and thealkylsilyl coating of Formula II can be(3-glycidyloxypropyl)trimethoxysilane followed by hydrolysis. In someembodiments, the alkylsilyl coating of Formula I isbis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane and thealkylsilyl coating of Formula II is n-decyltrichlorosilane. Thealkylsilyl coating of Formula I can be bis(trichlorosilyl)ethane orbis(trimethoxysilyl)ethane and the alkylsilyl coating of Formula II canbe trimethylchlorosilane or trimethyldimethyaminosilane. In someembodiments, the alkylsilyl coating of Formula I isbis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane and thealkylsilyl coating of Formula II is methoxy-polyethyleneoxy(3)silane.

The chromatographic device can also include an alkylsilyl coating havingthe Formula III in direct contact with the alkylsilyl coating of FormulaI,

R¹¹, R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are each independently selected from(C₁-C₆)alkoxy, —NH(C₁-C₆)alkyl, —N((C₁-C₆)alkyl)₂, OH, and halo. Z is(C₁-C₂₀)alkyl, —O[(CH₂)₂O]₁₋₂₀—, —(C₁-C₁₀)[NH(CO)NH(C₁-C₁₀)]₁₋₂₀-, or—(C₁-C₁₀)[alkylphenyl(C₁-C₁₀)alkyl]₁₋₂₀-. In some embodiments, thealkylsilyl coating of Formula III is bis(trichlorosilyl)ethane orbis(trimethoxysilyl)ethane. The alkylsilyl coating of Formula I can bebis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane and thealkylsilyl coating of Formula III can be bis(trichlorosilyl)ethane orbis(trimethoxysilyl)ethane. In some embodiments, the alkylsilyl coatingof I and III has a total thickness of about 400 Å.

In another aspect, the technology relates to a method of separating asample comprising a glycan, a peptide, or a pesticide. The methodincludes introducing the sample comprising the glycan, the peptide, orthe pesticide to a fluidic system including a flow path disposed in aninterior of the fluidic system. The flow path comprising a alkylsilylcoating having the Formula I:

R¹, R², R³, R⁴, R⁵, and R⁶ are each independently selected from(C₁-C₆)alkoxy, —NH(C₁-C₆)alkyl, —N((C₁-C₆)alkyl)₂, OH, OR^(A), and halo.R^(A) represents a point of attachment to the interior surfaces of thefluidic system. At least one of R¹, R², R³, R⁴, R⁵, and R⁶ is OR^(A). Xis (C₁-C₂₀)alkyl, —O[(CH₂)₂O]₁₋₂₀—, —(C₁-C₁₀)[NH(CO)NH(C₁-C₁₀)]₁₋₂₀-, or—(C₁-C₁₀)[alkylphenyl(C₁-C₁₀)alkyl]₁₋₂₀-. A second alkylsilyl coating isin direct contact with the alkylsilyl coating of Formula I. the secondalkylsilyl coating has the Formula II:

R⁷, R⁸, and R⁹ are each independently selected from —NH(C₁-C₆)alkyl,—N[(C₁-C₆)alkyl]₂, (C₁-C₆)alkoxy, (C₁-C₆)alkyl, (C₁-C₆)alkenyl, OH, andhalo. R¹⁰ is selected from (C₁-C₆)alkyl, —OR^(B),—[O(C₁-C₃)alkyl]₁₋₁₀O(C₁-C₆)alkyl, —[O(C₁-C₃)alkyl]₁₋₁₀OH, and phenyl.(C₁-C₆)alkyl is optionally substituted with one or more halo. The phenylis optionally substituted with one or more groups selected from(C₁-C₃)alkyl, hydroxyl, fluorine, chlorine, bromine, cyano, —C(O)NH₂,and carboxyl.

R^(B) is —(C₁-C₃)alkyloxirane, —(C₁-C₃)alkyl-3,4-epoxycyclohexyl, or—(C₁-C₄)alkylOH. The hashed bond to R¹⁰ represents an optionaladditional covalent bond between R¹⁰ and the carbon bridging the silylgroup to form an alkene, provided y is not 0. y is an integer from 0 to20. The method also includes eluting the sample through the fluidicsystem, thereby isolating the glycan, the peptide or the pesticide. Themethod can include one or more of the embodiments described herein inany combination thereof.

In some embodiments, the flow path is defined at least in part by theinterior surface of a chromatographic column. The flow path can befurther defined at least in part by passageways through a frit of thechromatographic column. In some embodiments, the flow path is furtherdefined at least in part by interior surfaces of tubing. The flow pathcan be formed of stainless steel

In some embodiments, the glycan is a phosphoglycan. The peptide can be aphosphopeptide. The pesticide can be glyphosate.

In some embodiments, the alkylsilyl coating of Formula I isbis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane. The alkylsilylcoating of Formula II can be n-decyltrichlorosilane. In someembodiments, the alkylsilyl coating of Formula I isbis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane and thealkylsilyl coating of Formula II is n-decyltrichlorosilane.

In another aspect, the technology includes a method of separating asample comprising a citric acid cycle metabolite. The method includesintroducing the sample comprising the citric acid cycle metabolite to afluidic system including a flow path disposed in an interior of thefluidic system. The flow path includes a alkylsilyl coating has theFormula I:

R¹, R², R³, R⁴, R⁵, and R⁶ are each independently selected from(C₁-C₆)alkoxy, —NH(C₁-C₆)alkyl, —N((C₁-C₆)alkyl)₂, OH, OR^(A), and halo.R^(A) represents a point of attachment to the interior surfaces of thefluidic system. At least one of R¹, R², R³, R⁴, R⁵, and R⁶ is OR^(A). Xis (C₁-C₂₀)alkyl, —O[(CH₂)₂O]₁₋₂₀—, —(C₁-C₁₀)[NH(CO)NH(C₁-C₁₀)]₁₋₂₀-, or—(C₁-C₁₀)[alkylphenyl(C₁-C₁₀)alkyl]₁₋₂₀-; A second alkylsilyl coating isin direct contact with the alkylsilyl coating of Formula I. The secondalkylsilyl coating has the Formula II:

R⁷, R⁸, and R⁹ are each independently selected from —NH(C₁-C₆)alkyl,—N[(C₁-C₆)alkyl]₂, (C₁-C₆)alkoxy, (C₁-C₆)alkyl, (C₁-C₆)alkenyl, OH, andhalo. R¹⁰ is selected from (C₁-C₆)alkyl, —OR^(B),—[O(C₁-C₃)alkyl]₁₋₁₀O(C₁-C₆)alkyl, —[O(C₁-C₃)alkyl]₁₋₁₀OH, and phenyl.The (C₁-C₆)alkyl is optionally substituted with one or more halo. Thephenyl is optionally substituted with one or more groups selected from(C₁-C₃)alkyl, hydroxyl, fluorine, chlorine, bromine, cyano, —C(O)NH₂,and carboxyl. R^(B) is —(C₁-C₃)alkyloxirane,—(C₁-C₃)alkyl-3,4-epoxycyclohexyl, or —(C₁-C₄)alkylOH. The hashed bondto R¹⁰ represents an optional additional covalent bond between R¹⁰ andthe carbon bridging the silyl group to form an alkene, provided y is not0. y is an integer from 0 to 20. The method also includes eluting thesample through the fluidic system, thereby isolating the citric acidcycle metabolite. The method can include one or more of the embodimentsdescribed herein in any combination thereof.

In some embodiments, the flow path is defined at least in part by theinterior surface of a chromatographic column. The flow path can befurther defined at least in part by passageways through a frit of thechromatographic column. The flow path can be further defined at least inpart by interior surfaces of tubing. The citric acid cycle metabolitecan be citric acid or malic acid.

In some embodiments, the alkylsilyl coating of Formula I isbis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane. The alkylsilylcoating of Formula II can be trimethylchlorosilane ortrimethyldimethyaminosilane. In some embodiments, the alkylsilyl coatingof Formula I is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethaneand the alkylsilyl coating of Formula II is trimethylchlorosilane ortrimethyldimethyaminosilane.

In another aspect, the technology includes a method of tailoring afluidic flow path for separation of a sample. The method includesassessing a polarity of an analyte in the sample, selecting achromatographic media based on the analyte in the sample, and selectinga alkylsilyl coating based on the polarity of the analyte in the sample.The alkylsilyl coating is inert to the analyte in the sample. Thealkylsilyl coating has the Formula I:

R¹, R², R³, R⁴, R⁵, and R⁶ are each independently selected from(C₁-C₆)alkoxy, —NH(C₁-C₆)alkyl, —N((C₁-C₆)alkyl)₂, OH, OR^(A), and halo.R^(A) represents a point of attachment to wetted surfaces of the fluidicflow path. At least one of R¹, R², R³, R⁴, R⁵, and R⁶ is OR^(A). X is(C₁-C₂₀)alkyl, —O[(CH₂)₂O]₁₋₂₀—, —(C₁-C₁₀)[NH(CO)NH(C₁-C₁₀)]₁₋₂₀-, or—(C₁-C₁₀)[alkylphenyl(C₁-C₁₀)alkyl]₁₋₂₀-. The method also includesadjusting a hydrophobicity of the wetted surfaces of the fluidic flowpath by vapor depositing the alkylsilyl coating onto the wetted surfacesof the fluidic flow path. The method can include one or more of theembodiments described herein in any combination thereof.

In some embodiments, the analyte in the sample is retained with aretentivity within 10% of the retentivity attributable to thechromatography media. In some embodiments, the analyte in the sample isretained with a retentivity within 5% of the retentivity attributable tothe chromatographic media. In some embodiments, the analyte in thesample is retained with a retentivity within 1% of the retentivityattributable to the chromatographic media.

The alkylsilyl coating can provide a desired contact angle of about 0°or about 95°.

In some embodiments, the wetted surface of the fluidic flow path aredefined at least in part by an interior surface of a column or aninterior surface of a sample injection needle. The wetted surface of thefluidic flow path can extend from an interior surface of a sampleinjection needle through the interior surface of a column. In someembodiments, the wetted surface of the fluidic flow path extends from asample reservoir container disposed upstream of an in fluidiccommunication with an interior surface of a sample injection needlethroughout the fluidic system to a connector or port of a detector.

The detector can be a mass spectrometer and the wetted surface of thefluidic flow path includes an interior surface of an electrosprayneedle.

In some embodiments, the alkylsilyl coating of Formula I isbis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane.

The method can also include annealing the alkylsilyl coating after vapordepositing the alkylsilyl coating onto the wetted surfaces of thefluidic flow path.

In some embodiments, the method also includes modifying the alkylsilylcoating of Formula I with a silanizing reagent to obtain a desiredthickness of the alkylsilyl coating. The silanizing reagent can be anon-volatile zwitterion. The non-volatile zwitterion can be sulfobetaineor carboxybetaine. The silianizing reagent can be an acidic or basicsilane. In some embodiments, the silanizing agent ismethoxy-polyethyleneoxy(6-9)silane.

In some embodiments, the method also includes adjusting thehydrophobicity of the wetted surfaces of the fluidic flow path by vapordepositing a second alkylsilyl coating in direct contact with the vapordeposited alkylsilyl coating of Formula I. The second alkylsilyl coatinghas the Formula II:

R⁷, R⁸, and R⁹ are each independently selected from —NH(C₁-C₆)alkyl,—N[(C₁-C₆)alkyl]₂, (C₁-C₆)alkoxy, (C₁-C₆)alkyl, (C₁-C₆)alkenyl, OH, andhalo. R¹⁰ is selected from (C₁-C₆)alkyl, —OR^(B),—[O(C₁-C₃)alkyl]₁₋₁₀O(C₁-C₆)alkyl, —[O(C₁-C₃)alkyl]₁₋₁₀OH, and phenyl,wherein said (C₁-C₆)alkyl is optionally substituted with one or morehalo and wherein said phenyl is optionally substituted with one or moregroups selected from (C₁-C₃)alkyl, hydroxyl, fluorine, chlorine,bromine, cyano, —C(O)NH₂, and carboxyl/R^(B) is —(C₁-C₃)alkyloxirane,—(C₁-C₃)alkyl-3,4-epoxycyclohexyl, or —(C₁-C₄)alkylOH. The hashed bondto R¹⁰ represents an optional additional covalent bond between R¹⁰ andthe carbon bridging the silyl group to form an alkene, provided y is not0. y is an integer from 0 to 20.

In some embodiments, the alkylsilyl coating of Formula II is(3-glycidyloxypropyl)trimethoxysilane, n-decyltrichlorosilane,trimethylchlorosilane, trimethyldimethyaminosilane, ormethoxy-polyethyleneoxy(3)silane. The alkylsilyl coating of Formula IIcan be (3-glycidyloxypropyl)trimethoxysilane followed by hydrolysis.

In some embodiments, the alkylsilyl coating of Formula I and II providesa desired contact angle of about 0° to about 105°.

The method can also include modifying the alkylsilyl coating of FormulaII with a silanizing reagent to obtain a desired thickness of thealkylsilyl coating. The silanizing reagent can be a non-volatilezwitterion. The non-volatile zwitterion can be sulfobetaine orcarboxybetaine. The silianizing reagent can be an acidic or basicsilane. In some embodiments, the silanizing agent ismethoxy-polyethyleneoxy(6-9)silane.

In some embodiments, the analyte is a biomolecule. The biomolecule is apeptide or peptide fragment, an oligopeptide, a protein, a glycan, anucleic acid or nucleic acid fragment, a growth factor, a carbohydrate,a fatty acid or a lipid. The analyte can be a citric acid cyclemetabolite. In some embodiments, the analyte is a pesticide.

The method can also include assessing a polarity of the chromatographicmedia and selecting the alkylsilyl coating based on the polarity of theanalyte and the chromatographic media.

In some embodiments, the also includes eluting the sample through thefluidic flow path, thereby isolating the analyte.

In another aspect, the technology includes a method of tailoring afluidic flow path for separation of a sample. The method includesinfiltrating a vaporized source of an alkylsilyl of Formula III onwetted surfaces of the fluidic flow path

R¹¹, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are each independently selected from(C₁-C₆)alkoxy, —NH(C₁-C₆)alkyl, —N((C₁-C₆)alkyl)₂, OH, and halo. Z is(C₁-C₂₀)alkyl, —O[(CH₂)₂O]₁₋₂₀—, —(C₁-C₁₀)[NH(CO)NH(C₁-C₁₀)]₁₋₂₀-, or—(C₁-C₁₀)[alkylphenyl(C₁-C₁₀)alkyl]₁₋₂₀-. The method also includescontrolling temperature and pressure to deposit a first coating ofFormula III on the wetted surfaces of the fluidic flow path, the firstcoating having a thickness of at least 100 Å and a contact angle of atleast 15°. The method can include one or more of the embodimentsdescribed herein in any combination thereof.

In some embodiments, the method also includes pretreating the wettedsurfaces of the fluidic flow path with a plasma prior to depositing thefirst coating of Formula III.

The method can also include modifying the alkylsilyl coating of FormulaIII with a silanizing reagent to obtain a desired thickness of thealkylsilyl coating. The silanizing reagent can be a non-volatilezwitterion. The non-volatile zwitterion can be sulfobetaine orcarboxybetaine. The silianizing reagent can be an acidic or basicsilane. In some embodiments, the silanizing agent ismethoxy-polyethyleneoxy(6-9)silane.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of a chromatographic flow system including achromatography column and various other components, in accordance withan illustrative embodiment of the technology. A fluid is carried throughthe chromatographic flow system with a fluidic flow path extending froma fluid manager to a detector.

FIG. 2 is a flow chart showing a method of tailoring wetted surfaces ofa flow path, in accordance with an illustrative embodiment of thetechnology.

FIG. 3 is a flow chart showing a method of tailoring a fluidic flow pathfor separation of a sample including a biomolecule, in accordance withan illustrative embodiment of the technology.

FIG. 4A shows a fluorescence chromatogram obtained using uncoatedstainless steel hardware, in accordance with an illustrative embodimentof the technology

FIG. 4B shows a fluorescence chromatogram obtained using hardware coatedwith exemplary vapor deposited alkylsilyl, in accordance with anillustrative embodiment of the technology.

FIG. 4C shows a fluorescence chromatogram obtained using hardware coatedwith exemplary vapor deposited alkylsilyl, in accordance with anillustrative embodiment of the technology.

FIG. 5A is a schematic of exemplified bioinert alkysilyl coatedstainless steel sample flow path components, including column inlettubing, in accordance with an illustrative embodiment of the technology.

FIG. 5B is a schematic of exemplified bioinert alkysilyl coatedstainless steel sample flow path components, including a sample needle,in accordance with an illustrative embodiment of the technology.

FIG. 6A shows a fluorescence chromatogram obtained using an untreatedflow path and untreated tube and frit combination in accordance with anembodiment of the technology.

FIG. 6B shows a fluorescence chromatogram obtained using an untreatedflow path and coated tube and frit combination, in accordance with anembodiment of the technology.

FIG. 6C shows a fluorescence chromatogram obtained using a coated flowpath and coated tube and frit combination, in accordance with anembodiment of the technology.

FIG. 7A shows a UV chromatogram obtained using an untreated stainlesssteel tube/frit combination in accordance with an embodiment of thetechnology.

FIG. 7B shows a UV chromatogram obtained using a C₂ vapor depositioncoated tube/frit combination, in accordance with an embodiment of thetechnology.

FIG. 7C shows a UV chromatogram obtained using a C₂C₁₀ vapor depositioncoated tube/frit combination, in accordance with an embodiment of thetechnology.

FIG. 8A is a chromatogram showing the effects of employing vapordeposition coated column hardware for the reversed phase LC analyses ofglucose-6-phosphate, in accordance with an illustrative embodiment ofthe technology.

FIG. 8B is a chromatogram showing the effects of employing untreatedcolumn hardware for the reversed phase LC analyses ofglucose-6-phosphate, in accordance with an illustrative embodiment ofthe technology.

FIG. 9A is a chromatogram showing the effects of employing vapordeposition coated column hardware for the reversed phase LC analyses offructose-6-phosphate, in accordance with an illustrative embodiment ofthe technology.

FIG. 9B is a chromatogram showing the effects of employing untreatedcolumn hardware for the reversed phase LC analyses offructose-6-phosphate, in accordance with an illustrative embodiment ofthe technology.

FIG. 10A is a chromatogram showing the effects of employing vapordeposition coated column hardware for the reversed phase LC analyses ofadenosine triphosphate, in accordance with an illustrative embodiment ofthe technology.

FIG. 10B is a chromatogram showing the effects of employing untreatedcolumn hardware for the reversed phase LC analyses of adenosinetriphosphate, in accordance with an illustrative embodiment of thetechnology.

FIG. 11A is a chromatogram showing the effects of employing vapordeposition coated column hardware for the reversed phase LC analyses ofadenosine monophosphate, in accordance with an illustrative embodimentof the technology.

FIG. 11B is a chromatogram showing the effects of employing untreatedcolumn hardware for the reversed phase LC analyses of adenosinemonophosphate, in accordance with an illustrative embodiment of thetechnology.

FIG. 12A is a fluorescence chromatogram for fetuin N-glycans obtainedwith untreated stainless steel, in accordance with an illustrativeembodiment of the technology.

FIG. 12B is a fluorescence chromatogram for fetuin N-glycans obtainedwith vapor deposition coated hardware, in accordance with anillustrative embodiment of the technology.

FIG. 13A is a graph showing fluorescence peak areas for disialyatedglycans obtained with untreated stainless steel hardware compared tostainless steel hardware coated different types of vapor depositedcoatings, in accordance with an illustrative embodiment of thetechnology.

FIG. 13B is a graph showing fluorescence peak areas for trisialyatedglycans obtained with untreated stainless steel hardware compared tostainless steel hardware coated different types of vapor depositedcoatings, in accordance with an illustrative embodiment of thetechnology.

FIG. 13C is a graph showing fluorescence peak areas for tetrasialyatedglycans obtained with untreated stainless steel hardware compared tostainless steel hardware coated different types of vapor depositedcoatings, in accordance with an illustrative embodiment of thetechnology.

FIG. 13D is a graph showing fluorescence peak areas for pentasialyatedglycans obtained with untreated stainless steel hardware compared tostainless steel hardware coated different types of vapor depositedcoatings, in accordance with an illustrative embodiment of thetechnology.

FIG. 14A is a reversed phase fluorescence chromatogram of reduced,IdeS-digested NIST Reference Material 8671 obtained with column hardwarecomponents treated with coatings in accordance with illustrativeembodiments of the technology.

FIG. 14B is a reversed phase fluorescence chromatogram of reduced,IdeS-digested NIST Reference Material 8671 obtained with column hardwarecomponents treated with coatings in accordance with illustrativeembodiments of the technology.

FIG. 15A is a reversed phase total ion chromatogram for columnsconstructed with stainless steel alternatives, namely polyether etherketone (PEEK) and a low titanium, nickel cobalt alloy (MP35NLT) withvarious components coated, in accordance with an illustrative embodimentof the technology.

FIG. 15B is a reversed phase total ion chromatogram for columncomponents constructed with stainless steel, C₂ coatings and C₂C₁₀coatings, in accordance with an illustrative embodiment of thetechnology.

FIG. 16A shows fluorescence chromatograms of reduced, IdeS-digested NISTReference Material 8671 and the effect on baseline return when variouscomponents of the system are coated, in accordance with an illustrativeembodiment of the technology.

FIG. 16B shows reversed phase total ion chromatograms (TICs) reduced,IdeS-digested NIST Reference Material 8671 and the effect on baselinereturn when various components of the system are coated, in accordancewith an illustrative embodiment of the technology.

FIG. 16C is a schematic of the column tube and frits that were coatedand used to obtain the chromatograms of FIGS. 16A and 16B, in accordancewith an illustrative embodiment of the technology.

FIG. 17A shows fluorescence chromatograms of reduced, IdeS-digested NISTReference Material 8671 and the effect on baseline return when variouscomponents of the system are coated, in accordance with an illustrativeembodiment of the technology.

FIG. 17B shows reversed phase total ion chromatograms (TICs) of reduced,IdeS-digested NIST Reference Material 8671 and the effect on baselinereturn when various components of the system are coated, in accordancewith an illustrative embodiment of the technology.

FIG. 17C is a schematic of the column tube and frits that were coatedand used to obtain the chromatograms of FIGS. 17A and 17B, in accordancewith an illustrative embodiment of the technology

FIG. 18 is a bar graph showing bubble point pressure in each of waterand IPA for a non-coated stainless steel frit and stainless steel fritscoated in accordance with one or more illustrative embodiments of thetechnology. The bubble point in water is provided as the left bar, andthe bubble point in IPA is provided as the right bar for each type offrit.

FIG. 19 is a bar graph showing a comparison of fit porosity contactangle with water for a non-coated stainless steel frit versus stainlesssteel frits coated in accordance with one or more embodiments of thetechnology.

FIG. 20 is a bar graph showing a comparison of mass loss test accordingto ASTM G48 Method A for a bare or uncoated stainless steel frit versusstainless steel frits coated in accordance with one or more embodimentsof the technology.

FIG. 21A is a chromatogram of NIST reference material 8671, an IgG1κmAb, as obtained from injection 1 using hardware A, uncoated, inaccordance with an illustrative embodiment of the technology.

FIG. 21B is a chromatogram of NIST reference material 8671, an IgG1κmAb, as obtained from injection 2 using hardware A, uncoated, inaccordance with an illustrative embodiment of the technology.

FIG. 21C is a chromatogram of NIST reference material 8671, an IgG1κmAb, as obtained from injection 3 using hardware A, uncoated, inaccordance with an illustrative embodiment of the technology.

FIG. 21D is a chromatogram of NIST reference material 8671, an IgG1κmAb, as obtained from injection 1 using hardware A, C₂ coating, inaccordance with an illustrative embodiment of the technology.

FIG. 21E is a chromatogram of NIST reference material 8671, an IgG1κmAb, as obtained from injection 2 using hardware A, C₂ coating, inaccordance with an illustrative embodiment of the technology.

FIG. 21F is a chromatogram of NIST reference material 8671, an IgG1κmAb, as obtained from injection 3 using hardware A, C₂ coating, inaccordance with an illustrative embodiment of the technology.

FIG. 21G is a chromatogram of NIST reference material 8671, an IgG1κmAb, as obtained from injection 1 using hardware B, uncoated, inaccordance with an illustrative embodiment of the technology.

FIG. 21H is a chromatogram of NIST reference material 8671, an IgG1κmAb, as obtained from injection 2 using hardware B, uncoated, inaccordance with an illustrative embodiment of the technology.

FIG. 21I is a chromatogram of NIST reference material 8671, an IgG1κmAb, as obtained from injection 3 using hardware B, uncoated, inaccordance with an illustrative embodiment of the technology.

FIG. 21J is a chromatogram of NIST reference material 8671, an IgG1κmAb, as obtained from injection 1 using hardware B, C₂-GPTMS-OH coating,in accordance with an illustrative embodiment of the technology.

FIG. 21K is a chromatogram of NIST reference material 8671, an IgG1κmAb, as obtained from injection 2 using hardware B, C₂-GPTMS-OH coating,in accordance with an illustrative embodiment of the technology.

FIG. 21L is a chromatogram of NIST reference material 8671, an IgG1κmAb, as obtained from injection 3 using hardware B, C₂-GPTMS-OH coating,in accordance with an illustrative embodiment of the technology.

FIG. 22 presents a bar graph showing peak areas of NIST referencematerials 8671 obtained from sequential cation exchange separations overthree injections of the sample, in accordance with an illustrativeembodiment of the technology. This bar graph compares the peak areas forfour different constructions in which the left most bar in eachinjection is an uncoated hardware A construction. The second from theleft is a coated version of hardware A. The third bar from the left isan uncoated hardware B construction and the fourth or last bar perinjection is a coated hardware B construction.

FIG. 23A is a reversed-phase chromatogram of the first injection of 5picomoles of deoxythymidine oligomers (15, 20, 25, 30, and 35-mer)obtained from a 2.1×50 mm 1.7 μm organosilica 130 Å C₁₈ columnconstructed with an untreated stainless steel (SS) tube and frits, inaccordance with an illustrative embodiment of the technology.

FIG. 23B is a reversed-phase chromatogram of the second injection of 5picomoles of deoxythymidine oligomers (15, 20, 25, 30, and 35-mer)obtained from a 2.1×50 mm 1.7 μm organosilica 130 Å C₁₈ columnconstructed with an untreated stainless steel (SS) tube and frits, inaccordance with an illustrative embodiment of the technology.

FIG. 23C is a reversed-phase chromatogram of the third injection of 5picomoles of deoxythymidine oligomers (15, 20, 25, 30, and 35-mer)obtained from a 2.1×50 mm 1.7 μm organosilica 130 Å C₁₈ columnconstructed with an untreated stainless steel (SS) tube and frits, inaccordance with an illustrative embodiment of the technology.

FIG. 23D is a reversed-phase chromatogram of the first injection of 5picomoles of deoxythymidine oligomers (15, 20, 25, 30, and 35-mer)obtained from a column constructed with a C₂C₁₀ vapor deposition coatedtube and frits, in accordance with an illustrative embodiment of thetechnology.

FIG. 23E is a reversed-phase chromatogram of the second injection of 5picomoles of deoxythymidine oligomers (15, 20, 25, 30, and 35-mer)obtained from a column constructed with a C₂C₁₀ vapor deposition coatedtube and frits, in accordance with an illustrative embodiment of thetechnology.

FIG. 23F is a reversed-phase chromatogram of the third injection of 5picomoles of deoxythymidine oligomers (15, 20, 25, 30, and 35-mer)obtained from a column constructed with a C₂C₁₀ vapor deposition coatedtube and frits, in accordance with an illustrative embodiment of thetechnology.

FIG. 24 is a graph showing the average UV peak areas of a 15-merdeoxythymidine analyte as observed during reversed phase chromatographyand initial injections onto either a 2.1×50 mm 1.7 μm organosilica 130 ÅC₁₈ column constructed with untreated stainless steel (SS) or C₂C₁₀vapor deposition coated components, in accordance with an illustrativeembodiment of the technology. Analyses were performed in duplicate usingtwo untreated columns and two C₂C₁₀ vapor deposition coated columns.

FIG. 25A is a reversed phase MRM (multiple reaction monitoring)chromatogram obtained for citric acid with the use of a 2.1×50 mm 1.8 μmsilica 100 Å C₁₈ 1.8 μm column constructed with C₂C₃ vapor depositioncoated components, in accordance with an illustrative embodiment of thetechnology.

FIG. 25B is a reversed phase MRM (multiple reaction monitoring)chromatogram obtained for citric acid with the use of a 2.1×50 mm 1.8 μmsilica 100 Å C₁₈ 1.8 μm column constructed with untreated components, inaccordance with an illustrative embodiment of the technology.

FIG. 25C is a reversed phase MRM (multiple reaction monitoring)chromatogram obtained for malic acid with the use of a 2.1×50 mm 1.8 μmsilica 100 Å C₁₈ 1.8 μm column constructed with C₂C₃ vapor depositioncoated components, in accordance with an illustrative embodiment of thetechnology.

FIG. 25D is a reversed phase MRM (multiple reaction monitoring)chromatogram obtained for malic acid with the use of a 2.1×50 mm 1.8 μmsilica 100 Å C₁₈ 1.8 μm column constructed with untreated components, inaccordance with an illustrative embodiment of the technology.

FIG. 26A is a mixed mode hydrophilic interaction chromatography (HILIC)MRM (multiple reaction monitoring) chromatogram of glyphosate showingMRM peak intensities obtained for glyphosate with the use of a 2.1×100mm 1.7 μm diethylamine bonded organosilica 130 Å column constructed withC₂C₁₀ vapor deposition coated components, in accordance with anillustrative embodiment of the technology.

FIG. 26B is a mixed mode hydrophilic interaction chromatography (HILIC)MRM (multiple reaction monitoring) chromatogram of glyphosate showingMRM peak intensities obtained for glyphosate with the use of a 2.1×100mm 1.7 μm diethylamine bonded organosilica 130 Å column with uncoatedcomponents, in accordance with an illustrative embodiment of thetechnology.

FIG. 27A is a graph showing the average peak areas of glyphosate asobserved during mixed mode HILIC using either a 2.1×100 mm 1.7 μmdiethylamine bonded organosilica 130 Å column constructed with eitheruntreated or C₂C₁₀ vapor deposition coated components in accordance withan illustrative embodiment of the technology. The analyses wereperformed with six replicate injections.

FIG. 27B is a graph showing the average peak widths of glyphosate asobserved during mixed mode HILIC using either a 2.1×100 mm 1.7 μmdiethylamine bonded organosilica 130 Å column constructed with eitheruntreated or C₂C₁₀ vapor deposition coated components, in accordancewith an illustrative embodiment of the technology. The analyses wereperformed with six replicate injections.

DETAILED DESCRIPTION

In general, a number of aspects of the technology are directed to (1)devices having an alkylsilyl coating; (2) methods of tailoring or tuninga flow path for isolation of an analyte, in particular ametal-interacting analyte; (3) method of isolating an analyte in asample, in particular a metal-interacting analyte; and (4) kitscomprising various chromatographic components coated with an alkylsilylcoating and instructions for use. In some aspects, a bioinert, low-bindcoating is used to modify a flow path to address flow path interactionswith an analyte. That is, the bioinert, low-bind coating minimizessurface reactions with the metal interacting analyte and allows theanalyte to pass along a flow path without clogging, attaching tosurfaces, or change in analyte properties. The reduction/elimination ofthese interactions is advantageous because it allows for accuratequantification and analysis of a sample containing a metal-interactinganalyte, for example biomolecules, proteins, glycans, peptides,oligonucleotides, pesticides, bisphosphonic acids, anionic metabolites,and zwitterions like amino acids and neurotransmitters. The biomoleculecan be selected from a peptide or peptide fragment, an oligopeptide, aprotein, a glycan, a nucleic acid or nucleic acid fragment, a growthfactor, a carbohydrate, a fatty acid, and a lipid. In one aspect, thebiomolecule is a protein, a peptide, or a glycan. The biomolecule can bea phosphoglycan or a phosphopeptide.

In the present technology, vapor deposited alkylsilyl coatings on wettedsurfaces of fluidic systems (e.g., liquid chromatography systems) modifythe fluidic path and decrease secondary interactions. As such, they arebioinert or low-bind (meaning that analytes of a sample do not interact,or have minimal interaction, with the alkylsilyl coating). In addition,the deposited coatings are highly tunable to provide a range ofdesirable contact angles (e.g., make the wetted surfaces hydrophilic orhydrophobic), chemistries, and properties to achieve a desired effect onthe flow path and ultimately the sample passing through the flow path.

Devices

FIG. 1 is a representative schematic of a chromatographic flowsystem/device 100 that can be used to separate analytes in a sample.Chromatographic flow system 100 includes several components including afluid manager system 105 (e.g., controls mobile phase flow through thesystem), tubing 110 (which could also be replaced or used together withmicrofabricated fluid conduits), fluid connectors 115 (e.g., fluidiccaps), frits 120, a chromatography column 125, a sample injector 135including a needle (not shown) to insert or inject the sample into themobile phase, a vial, sinker, or sample reservoir 130 for holding thesample prior to injection, a detector 150 and a pressure regulator 140for controlling pressure of the flow. Interior surfaces of thecomponents of the chromatographic system/device form a fluidic flow paththat has wetted surfaces. The fluidic flow path can have a length todiameter ratio of at least 20, at least 25, at least 30, at least 35 orat least 40.

The detector 150, can be a mass spectrometer. The fluidic flow path caninclude wetted surfaces of an electrospray needle (not shown).

At least a portion of the wetted surfaces can be coated with an alkylsilyl coating, described in detail herein, to tailor its hydrophobicity.The coating can be applied by vapor deposition. As such, methods anddevices of the present technology provide the advantage of being able touse high pressure resistant materials (e.g., stainless steel) for thecreation of the flow system, but also being able to tailor the wettedsurfaces of the fluidic flow path to provide the appropriatehydrophobicity so deleterious interactions or undesirable chemicaleffects on the sample can be minimized.

The alkylsilyl coating can be provided throughout the system from thetubing or fluid conduits 110 extending from the fluid manager system 105all the way through to the detector 150. The coatings can also beapplied to portions of the fluidic fluid path. That is, one may chooseto coat one or more components or portions of a component and not theentire fluidic path. For example, the internal portions of the column125 and its frits 120 and end caps 115 can be coated whereas theremainder of the flow path can be left unmodified. Further,removable/replaceable components can be coated. For example, the vial orsinker 130 containing the sample reservoir can be coated as well asfrits 120.

In one aspect, the flow path of the fluidic systems described herein isdefined at least in part by an interior surface of tubing. In anotheraspect, the flow path of the fluidic systems described herein is definedat least in part by an interior surface of microfabricated fluidconduits. In another aspect, the flow path of the fluidic systemsdescribed herein is defined at least in part by an interior surface of acolumn. In another aspect, the flow path of the fluidic systemsdescribed herein is defined at least in part by passageways through afrit. In another aspect, the flow path of the fluidic systems describedherein is defined at least in part by an interior surface of a sampleinjection needle. In another aspect, the flow path of the fluidicsystems described herein extends from the interior surface of a sampleinjection needle throughout the interior surface of a column. In anotheraspect, the flow path extends from a sample reservoir container (e.g.sinker) disposed upstream of and in fluidic communication with theinterior surface of a sample injection needle throughout the fluidicsystem to a connector/port to a detector.

In some embodiments, only the wetted surfaces of the chromatographiccolumn and the components located upstream of the chromatographic columnare coated with the alkylsilyl coatings described herein while wettedsurfaces located downstream of the column are not coated. The coatingcan be applied to the wetted surfaces via vapor deposition.

At least a portion of the wetted surfaces of the fluidic flow path arecoated with an alkylsilyl coating. The alkylsilyl coating is inert to atleast one of the analytes in the sample. The alkylsilyl coating can havethe Formula I:

R¹, R², R³, R⁴, R⁵, and R⁶ are each independently selected from(C₁-C₆)alkoxy, —NH(C₁-C₆)alkyl, —N((C₁-C₆)alkyl)₂, OH, OR^(A), and halo(i.e., a halogen, for example chloro). R^(A) represents a point ofattachment to the interior surfaces of the fluidic system. At least oneof R¹, R², R³, R⁴, R⁵, and R⁶ is OR^(A). X is (C₁-C₂₀)alklyl,—O[(CH₂)₂O]₁₋₂₀—, —(C₁-C₁₀)[NH(CO)NH(C₁-C₁₀)]₁₋₂₀-, or —(C₁-C₁₀)[alkylphenyl(C₁-C₁₀)alkyl]₁₋₂₀-.

When used in the context of a chemical formula, a hyphen (“-”) indicatesthe point of attachment. For example, when X is—[(C₁-C₁₀)alkylphenyl(C₁-C₁₀)alkyl]₁₋₂₀-, that means that X is connectedto SiR¹R²R³ via the (C₁-C₁₀)alkyl and connected to SiR⁴R⁵R⁶ via theother (C₁-C₁₀)alkyl. This applies to the remaining variables.

In one aspect, X in Formula I is (C₁-C₁₅)alkyl, (C₁-C₁₂)alkyl, or(C₁-C₁₀)alkyl. In some aspects, X in Formula I is methyl, ethyl, propyl,isopropyl, butyl, sec-butyl, iso-butyl, t-butyl, pentyl, hexyl, heptyl,nonyl, or decanyl. In other aspect, X in Formula I is ethyl or decanyl.

In one aspect, at least one of R¹, R², R³, R⁴, R⁵, and R⁶ is(C₁-C₆)alkoxy, e.g., ethoxy, wherein the values for X are described inFormula I or the preceding paragraph. In another aspect, at least two ofR¹, R², R³, R⁴, R⁵, and R⁶ is (C₁-C₆)alkoxy, e.g., ethoxy, wherein thevalues for X are described in Formula I or the preceding paragraph. Inanother aspect, at least three of R¹, R², R³, R⁴, R⁵, and R⁶ is(C₁-C₆)alkoxy, e.g., ethoxy, wherein the values for X are described inFormula I or the preceding paragraph. In another aspect, at least fourof R¹, R², R³, R⁴, R⁵, and R⁶ is (C₁-C₆)alkoxy, e.g., ethoxy, whereinthe values for X are described in Formula I or the preceding paragraph.In another aspect, at least five of R¹, R², R³, R⁴, R⁵, and R⁶ is(C₁-C₆)alkoxy, e.g., ethoxy, wherein the values for X are described inFormula I or the preceding paragraph.

In one aspect, at least one of R¹, R², R³, R⁴, R⁵, and R⁶ is halo, e.g.,chloro, wherein the values for X are described in Formula I or thepreceding paragraphs above. In another aspect, at least two of R¹, R²,R³, R⁴, R⁵, and R⁶ is halo, e.g., chloro, wherein the values for X aredescribed in Formula I or the preceding paragraphs above. In anotheraspect, at least three of R¹, R², R³, R⁴, R⁵, and R⁶ is halo, e.g.,chloro, wherein the values for X are described in Formula I or thepreceding paragraphs above. In another aspect, at least four of R¹, R²,R³, R⁴, R⁵, and R⁶ is halo, e.g., chloro, wherein the values for X aredescribed in Formula I or the preceding paragraphs above. In anotheraspect, at least five of R¹, R², R³, R⁴, R⁵, and R⁶ is halo, e.g.,chloro, wherein the values for X are described in Formula I or thepreceding paragraphs above.

In another aspect, R¹, R², R³, R⁴, R⁵, and R⁶ are each methoxy orchloro.

The alkylsilyl coating of Formula I can have a contact angle of at leastabout 15°. In some embodiments, the alkylsilyl coating of Formula I canhave a contact angle of less than or equal to 30°. The contact angle canbe less than or equal to about 90°. In some embodiments, the contactangle of the alkylsilyl coating of Formula I is between about 15° toabout 105°. For example, the contact angle of the alkylsilyl coating ofFormula I can be about 0°, 5°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°,50°, 55°, 60°, 65°, 70°, 75°, 80°, 85°, 90°, 95°, 100°, or 105°.

The thickness of the alkylsilyl coating can be at least about 100 Å. Forexample the thickness can be between about 100 Å to about 1600 Å. Thethickness of the alkylsilyl coating for Formal I can be about 100 Å, 200Å, 300 Å, 400 Å, 500 Å, 600 Å, 700 Å, 800 Å, 900 Å, 1000 Å, 1100 Å, 1200Å, 1300 Å, 1400 Å, 1500 Å or 1600 Å. The thickness of the alkylsilylcoating (e.g., a vapor deposited alkylsilyl coating) can be detectedoptically by the naked eye. For example, more opaqueness and colorationis indicative of a thicker coating. Thus, coatings with pronouncedvisual distinction are an embodiment of this technology. From thin tothick, the color changes from yellow, to violet, to blue, to slightlygreenish and then back to yellow when coated parts are observed underfull-spectrum light, such as sunlight. For example, when the alkylsilylcoating is 300 Å thick, the coating can appear yellow and reflect lightwith a peak wavelength between 560 and 590 nm. When the alkylsilylcoating is 600 Å thick, the coating can appear violet and reflect lightwith a peak wavelength between 400 and 450 nm. When the alkylsilylcoating is 1000 Å thick, the coating can appear blue and reflect lightwith a peak wavelength between 450 and 490 nm. See, e.g., Faucheu etal., Relating Gloss Loss to Topographical Features of a PVDF Coating,Published Oct. 6, 2004; Bohlin, Erik, Surface and Porous Structure ofPigment Coatings, Interactions with flexographic ink and effects ofprint quality, Dissertation, Karlstad University Studies, 2013:49.

In one aspect, the vapor deposited coating of Formula I is the productof vapor deposited bis(trichlorosilyl)ethane,bis(trimethoxysilyl)ethane, bis(trichlorosilyl)octane,bis(trimethoxysilyl)octane, bis(trimethoxysilyl)hexane, andbis(trichlorosilyl)hexane.

In some aspects, at least a portion of the wetted surfaces of thefluidic flow path are coated with multiple layers of the same ordifferent alkysilyls, where the thickness of the alkylsilyl coatingscorrelate with the number of layering steps performed (e.g., the numberof deposited layers of alkylsilyl coating on wetted surface of thefluidic flow path of the chromatographic system/device). In this manner,increasingly thick bioinert coatings can be produced and tailored toachieve desirable separations.

The chromatographic device can have a second alkylsilyl coating indirect contact with the alkylsilyl coating of Formula I. The secondalkylsilyl coating has the Formula II

wherein R⁷, R⁸, and R⁹ are each independently selected from—NH(C₁-C₆)alkyl, —N[(C₁-C₆)alkyl]₂, (C₁-C₆)alkoxy, (C₁-C₆)alkyl,(C₁-C₆)alkenyl, OH, and halo; R¹⁰ is selected from (C₁-C₆)alkyl,—OR^(B), [O(C₁-C₃)alkyl]₁₋₁₀O(C₁-C₆)alkyl, —[O(C₁-C₃)alkyl]₁₋₁₀OH andphenyl. (C₁-C₆)alkyl is optionally substituted with one or more halo.The phenyl is optionally substituted with one or more groups selectedfrom (C₁-C₃)alkyl, hydroxyl, fluorine, chlorine, bromine, cyano,—C(O)NH₂, and carboxyl. R^(B) is —(C₁-C₃)alkyloxirane,—(C₁-C₃)alkyl-3,4-epoxycyclohexyl, or —(C₁-C₄)alkylOH. The hashed bondto R¹⁰ represents an optional additional covalent bond between R¹⁰ andthe carbon bridging the silyl group to form an alkene, provided y is not0. y is an integer from 0 to 20.

In one aspect, y in Formula II is an integer from 1 to 15. In anotheraspect, y in Formula II is an integer from 1 to 12. In another aspect, yin Formula II is an integer from 1 to 10. In another aspect, y inFormula II is an integer from 2 to 9.

In one aspect R¹⁰ in Formula II is methyl and y is as described abovefor Formula II or the preceding paragraph.

In one aspect, R⁷, R⁸, and R⁹ in Formula II are each the same, whereinR¹⁰ and y are as described above. In one aspect, R⁷, R⁸, and R⁹ are eachhalo (e.g., chloro) or (C₁-C₆)alkoxy such as methoxy, wherein R¹⁰ and yare as described above.

In one aspect, y in Formula II is 9, R¹⁰ is methyl, and R⁷, R⁸, and R⁹are each ethoxy or chloro.

In one aspect, the coating of the formula II is n-decyltrichlorosilane,(3-glycidyloxypropyl)trimethoxysilane (GPTMS),(3-glycidyloxypropyl)trimethoxysilane (GPTMS) followed by hydrolysis,2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, trimethylchlorosilane,trimethyldimethyaminosilane, methoxy-polyethyleneoxy(3)silanepropyltrichlorosilane, propyltrimethoxysilane,(heptadecafluoro-1,1,2,2-tetrahydrodecyl)tris(dimethylamino)silane,(heptadecafluoro-1,1,2,2-tetrahydrodecyl)trischlorosilane,(heptadecafluoro-1,1,2,2-tetrahydrodecyl)trimethoxysilanevinyltrichlorosilane, vinyltrimethoxysilane, allyltrichlorosilane,2-[methoxy(polyethyleneoxy)3propyl]trichlorosilane,2-[methoxy(polyethyleneoxy)3propyl]trimethoxysilane, or2-[methoxy(polyethyleneoxy)3propyl]tris(dimethylamino)silane.

The alkylsilyl coating of Formula I and II can have a contact angle ofat least about 15°. In some embodiments, the alkylsilyl coating ofFormula I and II can have a contact angle of less than or equal to 105°.The contact angle can be less than or equal to about 90°. In someembodiments, the contact angle of the alkylsilyl coating of Formula Iand II is between about 15° to about 105°. For example, the contactangle of the alkylsilyl coating of Formula I and II can be about 0°, 5°,10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°,80°, 85°, 90°, 95°, 100°, or 105°.

The thickness of the multi-layered alkylsilyl coating can be at leastabout 100 Å. For example the thickness can be between about 100 Å toabout 1600 Å. The thickness of the multi-layered alkylsilyl coating forFormal I can be about 100 Å, 200 Å, 300 Å, 400 Å, 500 Å, 600 Å, 700 Å,800 Å, 900 Å, 1000 Å, 1100 Å, 1200 Å, 1300 Å, 1400 Å, 1500 Å or 1600 Å.

In one aspect, the alkylsilyl coating of Formula I isbis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane and thealkylsilyl coating of Formula II is(3-glycidyloxypropyl)trimethoxysilane. In another aspect, the alkylsilylcoating of Formula I is bis(trichlorosilyl)ethane orbis(trimethoxysilyl)ethane and the alkylsilyl coating of Formula II is(3-glycidyloxypropyl)trimethoxysilane followed by hydrolysis. In oneaspect, the alkylsilyl coating of Formula I is bis(trichlorosilyl)ethaneor bis(trimethoxysilyl)ethane and the alkylsilyl coating of Formula IIis n-decyltrichlorosilane. The alkylsilyl coating of Formula I can bebis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane and thealkylsilyl coating of Formula II can be trimethylchlorosilane ortrimethyldimethyaminosilane. In one aspect, the alkylsilyl coating ofFormula I is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane andthe alkylsilyl coating of Formula II is methoxy-polyethyleneoxy(3)propyl tricholorosilane or methoxy-polyethyleneoxy(3) propyltrimethoxysilane.

The chromatographic device can have an alkylsilyl coating in directcontact with the alkylsilyl coating of Formula III in direct contactwith the alkylsilyl coating of Formula I.

R¹¹, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are each independently selected from(C₁-C₆)alkoxy, —NH(C₁-C₆)alkyl, —N((C₁-C₆)alkyl)₂, OH, and halo (i.e., ahalogen, for example, chloro). Z is (C₁-C₂₀)alkyl, —O[(CH₂)₂O]₁₋₂₀—,—(C₁-C₁₀)[NH(CO)NH(C₁-C₁₀)]₁₋₂₀-, or—(C₁-C₁₀)[alkylphenyl(C₁-C₁₀)alkyl]₁₋₂₀-. In some aspects, Z in FormulaIII is (C₁-C₁₀)alkyl; and R¹, R², R³, R⁴, R⁵, and R⁶ are each methoxy orchloro. In other aspects, Z in Formula III is (C₂-C₁₀)alkyl. In otheraspects, Z in Formula III is ethyl.

In the layered alkylsilyl coating of Formula I and Formula III, FormulaI and Formula III can be the same (for example, C₂C₂) or Formula I andFormula III can be different. Formula III is attached directly to thecoating of Formula I, i.e., in Formula III, there is no point ofattachment to the interior of the fluidic system; instead Formula III isdeposited directly on Formula I.

The alkylsilyl coating of Formula III can be bis(trichlorosilyl)ethaneor bis(trimethoxysilyl)ethane. The alkylsilyl coating of Formula I canbe bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane and thealkylsilyl coating of Formula III can be bis(trichlorosilyl)ethane orbis(trimethoxysilyl)ethane.

The alkylsilyl coating of Formula I and III can have a contact angle ofat least about 15°. In some embodiments, the alkylsilyl coating ofFormula I and III can have a contact angle of less than or equal to105°. The contact angle can be less than or equal to about 90°. In someembodiments, the contact angle of the alkylsilyl coating of Formula Iand III is between about 15° to about 105°. For example, the contactangle of the alkylsilyl coating of Formula I and III can be about 0°,5°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°,75°, 80°, 85°, 90°, 95°, 100°, or 105°.

The thickness of the multi-layered alkylsilyl coating can be at leastabout 100 Å. For example the thickness can be between about 100 Å toabout 1600 Å. The thickness of the multi-layered alkylsilyl coating forFormal I can be about 100 Å, 200 Å, 300 Å, 400 Å, 500 Å, 600 Å, 700 Å,800 Å, 900 Å, 1000 Å, 1100 Å, 1200 Å, 1300 Å, 1400 Å, 1500 Å or 1600 Å.

In one aspect, the alkylsilyl coating of Formula II is applied directlyto wetted surfaces of the fluidic flow path. Therefore, in someembodiments, one of R⁷, R⁸, and R⁹ of Formula II can also includeOR^(A), where R^(A) represents a point of attachment to the interiorsurfaces (e.g., wetted surfaces) of the fluidic system. In otherembodiments, R⁷, R⁸, and R⁹ of the alkylsilyl coating of Formula II doesnot include OR^(A), by the alkylsilyl coating of Formula II is depositeddirectly onto wetted surfaces of the fluidic flow path that have beenpre-treated with, for example, a plasma.

In one aspect, stainless steel flow path components, including but notlimited to tubing, microfabricated fluid conduits, column frits, columninlet tubing, and sample injection needles, are coated via vapordeposition with one or more of the disclosed alkylsilyls. In one aspect,these coated components are annealed to alter their chemical or physicalproperties.

In another aspect, flow path components that are made of other materialsthan stainless steel or other metallics, (e.g., polymers, glass, etc.)are coated via vapor deposition with one or more of the disclosedalkylsilyls. In particular, frits for use within the system or samplevials connectable to the injection needle are coated.

Exemplary coatings with their respective approximate thickness andcontact angle are provided in Table 1.

TABLE 1 Alternative Approximate Approximate Coating Thickness of ContactVPD# Vapor Deposited Material Abbreviation Product Angle 1bis(trichlorosilyl)ethane or C₂-GPTMS-OH 500 Å 15°bis(trismethoxysilyl)ethane as a first layer followed by GPTMS followedby hydrolysis to form GPTMS-OH 2 bis(trichlorosilyl)ethane or C₂ 500 Å35° bis(trimethoxysilyl)ethane 3 bis(trichlorosilyl)ethane or C₂-C₂ 1600Å  35° bis(trimethoxysilyl)ethane as a first layer followed bybis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane as a secondlayer. 4 bis(trichlorosilyl)ethane or C₂-GPTMS 500 Å 50°bis(trimethoxysilyl)ethane as a first layer followed by GPTMS as asecond layer 5 Annealed Annealed C₂ 500 Å 95° bis(trichlorosilyl)ethaneor bis(trimethoxysilyl)ethane 6 Annealed Annealed 1600 Å  95°bis(trichlorosilyl)ethane or C₂-C₂ bis(trimethoxysilyl)ethane as a firstlayer followed by annealed bis(trichlorosilyl)ethane orbis(trimethoxysilyl)ethane as a second layer 7 bis(trichlorosilyl)ethaneor C₂C₁₀ 500 Å 105° bis(trimethoxysilyl)ethane as a first layer followedby n- decyltrichlorosilane as a second layer 8 Annealed Annealed 500 Å105° bis(trichlorosilyl)ethane or C₂C₁₀ bis(trimethoxysilyl)ethane as afirst layer followed by annealed n-decyltrichlorosilane as a secondlayer 9 GPTMS GPTMS 100 to 200 Å     ~50° 10 GPTMS followed byhydrolysis GPTMS-OH 100 to 200 Å     ~20° to form GPTMS-OH 11bis(trichlorosilyl)ethane or C₂C₃ 500 Å 40-90°bis(trimethoxysilyl)ethane as a first layer followed bytrimethylchlorosilane or trimethyldimethylaminosilane 12 annealedAnnealed 500 Å 95° bis(trichlorosilyl)ethane or C₂C₃bis(trimethoxysilyl)ethane as a first layer followed bytrimethylchlorosilane or trimethyldimethylaminosilane 13bis(trichlorosilyl)ethane or C₂PEO 500 Å 15° bis(trimethoxysilyl)ethaneas a first layer followed by a methoxy-polyethyleneoxy(3) propyltrichlorosilane or methoxy-polyethyleneoxy(3) propyl trimethoxysilane

Referring to VPD #1 (C₂-GPTMS-OH), the first coating layer, C₂ shownbelow, is a layer according to Formula I, described above.

The second layer of VPD #1, GPTMS-OH, shown below, is a layer accordingto Formula II.

VPD #3 (C₂-C₂) is an example of a coating of Formula I and then acoating for Formula III.

VPD#7 (C₂C₁₀) is another example of a coating of Formula I and a secondlayer of Formula II. The structure of bis(trichlorosilyl)ethane orbis(trismethoxysilyl)ethane (C₂) is shown above. The structure of C₁₀ isshown below.

VPD #11 (C₂C₃) is another example of a coating of Formula I and a secondlayer of Formula II. The structure of bis(trichlorosilyl)ethane orbis(trismethoxysilyl)ethane (C₂) is shown above. The structure of C₃ isshown below.

VPD #13 is another example of a coating of Formula I and a second layerof Formula II. The structure of bis(trichlorosilyl)ethane orbis(trismethoxysilyl)ethane (C₂) is shown above. The structure ofmethoxy-polyethyleneoxy(3)propyl trichlorosilane (PEO) is shown below.

Alternatively, commercially available vapor deposition coatings can beused in the disclosed systems, devices, and methods, including but notlimited to Dursan® (commercially available from SikcoTek Corporation,Bellefonte, Pa.).

In one aspect, the alkylsilyl coatings described herein enhance thecorrosion performance of metals, e.g., as in metallic chromatographycolumns. Depending on the denseness and thickness, the coatings act as abarrier, thereby preventing water and corrosive molecules from reactingwith the base metal. While increasing the hydrophobicity and densityimproves the corrosion performance, even coatings derived from C₂ andGPTMS (C₂-GPTMS) followed by hydrolysis to form C₂-GPTMS-OH shows a 10×improvement in the ASTM G48 Method A pitting corrosion, see e.g.,Example 4 below. In terms of most corrosion resistant to least, theranking is the material formed from VPD#7>2>1 (bis(trichlorosilyl)ethaneor bis(trimethoxysilyl)ethane as a first layer followed by GPTMS thenhydrolysis to form GPTMS-OH as a second layer). This also correlates tohydrophobicity rankings.

Methods of Tailoring a Fluidic Flow Path

The coatings described above can be used to tailor a fluidic flow pathof a chromatography system for the separation of a sample. The coatingscan be vapor deposited. In general, the deposited coatings can be usedto adjust the hydrophobicity of internal surfaces of the fluidic flowpath that come into contact with a fluid (i.e. wetted surfaces orsurfaces coming into contact with the mobile phase and/orsample/analyte). By coating wetted surfaces of one or more components ofa flow path within a chromatography system, a user can tailor the wettedsurfaces to provide a desired interaction (or lack of interaction)between the flow path and fluids therein (including any sample, such asbiomolecules, proteins, glycans, peptides, oligonucleotides, pesticides,bisphosphonic acids, anionic metabolites, and zwitterions like aminoacids and neurotransmitters, within the fluid).

In one aspect, an effective coating is produced from a low temperature,vacuum-assisted vapor deposition process. In one aspect, an oxygenplasma pretreatment step precedes the coating deposition. The oxygenplasma removes organic compounds and improves surface wettability forthe coatings. Time, temperature, and pressure are controlled for eachprocessing step. Each coating run can use a silicon wafer to monitor thethickness and contact angle of the resultant coating. Ellipsometry canbe used to measure the coating thickness, and an optical goniometer canbe used to measure the contact angle of the coating. A post coatingannealing step can be utilized to increase coating cross-linking andincrease coating hydrophobicity.

FIG. 2 is a flow chart illustrating method 200 for tailoring a fluidicflow path for separation of a sample including biomolecules, proteins,glycans, peptides, oligonucleotides, pesticides, bisphosphonic acids,anionic metabolites, and zwitterions like amino acids andneurotransmitters. The method has certain steps which are optional asindicated by the dashed outline surrounding a particular step. Method200 can start with a pretreatment step (205) for cleaning and/orpreparing a flow path within a component for tailoring. Pretreatmentstep 205 can include cleaning the flow path with plasma, such as oxygenplasma. This pretreatment step is optional.

Next, an infiltration step (210) is initiated. A vaporized source of analkylsilyl compound (e.g., the alkylsilyl compounds of Formulas I, IIand/or III) is infiltrated into the flow path. The vaporized source isfree to travel throughout and along the internal surfaces of the flowpath. Temperature and/or pressure is controlled during infiltration suchthat the vaporized source is allowed to permeate throughout the internalflow path and to deposit a coating from the vaporized source on theexposed surface (e.g., wetted surfaces) of the flow path as shown instep 215. Additional steps can be taken to further tailor the flow path.For example, after the coating is deposited, it can be heat treated orannealed (step 220) to create cross linking within the deposited coatingand/or to adjust the contact angle or hydrophobicity of the coating.Additionally or alternatively, a second coating of alkylsilyl compound(having the same or different form) can be deposited by infiltrating avaporized source into the flow path and depositing a second oradditional layers in contact with the first deposited layer as shown instep 225. After the deposition of each coating layer, an annealing stepcan occur. Numerous infiltration and annealing steps can be provided totailor the flow path accordingly (step 230).

FIG. 3 provides a flow chart illustrating a method (300) of tailoring afluidic flow path for separation of a sample including a biomolecule ora metal interacting analyte. The method can be used to tailor a flowsystem for use in isolating, separating, and/or analyzing thebiomolecule or metal interacting analyte. In step 305, the analyte isassessed to determine its polarity. Understanding the polarity willallow an operator to select (by either look up table or make adetermination) a desired coating chemistry and, optionally, contactangle as shown in step 310. In some embodiments, in addition toassessing the polarity of the biomolecule or metal interacting analyte,the polarity of a stationary phase to be used to separate thebiomolecule or metal interacting analyte (e.g., stationary phase to beincluded in at least a portion of the fluidic flow path) is alsoassessed. A chromatographic media can be selected based on the analytein the sample. Understanding the polarity of both the analyte and thestationary phase is used in certain embodiments, by the operator toselect the desired coating chemistry and contact angle in step 310. Thecomponents to be tailored can then be positioned within a chemicalinfiltration system with environmental control (e.g., pressure,atmosphere, temperature, etc.) and precursor materials are infiltratedinto the flow path of the component to deposit one or more coatingsalong the wetted surfaces to adjust the hydrophobicity as shown in step315. During any one of infiltration, deposition, and condition steps(e.g. annealing), coatings deposited from the infiltration system can bemonitored and if necessary precursors and or depositing conditions canbe adjusted if required allowing for fine tuning of coating properties.The alkylsilyl coating material selected in step 310 can be thealkylsilyl compounds of Formulas I, II and/or III.

A method of tailoring a fluidic flow path for separation of a sample isprovided that includes assessing a polarity of an analyte in the sampleand selecting a chromatographic media based on the analyte in thesample. An alkylsilyl coating is selected based on the polarity of theanalyte in the sample. The alkylsilyl coating is selected so that thecoating is inert to the analyte(s) being separated. In other words, thealkylsilyl coating does not produce any secondary chromatographiceffects that are attributable to the alkylsilyl coating. In someembodiments, the analyte is a biomolecule. The biomolecule can be apeptide or peptide fragment, an oligopeptide, a protein, a glycan, anucleic acid or nucleic acid fragment, a growth factor, a carbohydrate,a fatty acid or a lipid. The analyte can be a citric acid cyclemetabolite. The analyte can be a pesticide.

The alkylsilyl coating can have the Formula I, II, or III as describedabove. In one embodiment, the alkylsilyl coating has the Formula I as afirst layer and Formula II as a second layer. In some embodiments, thereis only a single layer coating having Formula I (e.g.,bis(trichlorosilyl)ethane or bis(trimethoxysilyl)eithane). In someembodiments, there is only a single layer coating having Formula II(e.g., (3-glycidyloxypropyl)trimethoxysilane, n-decyltrichlorosilane,trimethylchlorosilane, trimethyldimethyaminosilane, ormethoxy-polyethyleneoxy(3)silane). In some embodiments, there is only asingle layer coating having Formula III (e.g., bis(trichlorosilyl)ethaneor bis(trimethoxysilyl)eithane).

The method also includes adjusting a hydrophobicity of the wettedsurfaces of the fluidic flow path by vapor depositing the alkylsilylcoating onto the wetted surfaces of the fluidic flow path. In someembodiments, the hydrophobicity of the wetted surfaces is adjusted byadjusting the contact angle of the alkylsilyl coating. For example, thecontact angle of the alkylsilyl coating can be between about 0° to about105°.

The analyte in the sample can be retained with a retentivity within 10%of the retentivity attributable to the chromatography media. In someembodiments, the sample can be retained with a retentivity within 5% orwithin 1% of the retentivity attributable to the chromatography media.Therefore, the alkylsilyl coating solves the problem of metalinteraction between the analyte and the metal chromatographic materialswithout introducing any secondary reactions that would have a negativeeffect on the quality of the separation. The alkylsilyl coating does notimpart any retention mechanism on the analyte of interest, making thecoating inert to the analyte of interest and low-bind.

In addition, the alkylsilyl coating does not produce any changes to peakwidth. The analyte in the sample has a peak width that is within 10%,5%, or 1% of the peak width attributable to the chromatographic media.

The wetted surfaces of the fluidic flow path can be any of thosedescribed above with respect to aspects and embodiments of thechromatographic device.

The method can also include annealing the alkylsilyl coating after vapordepositing the alkylsilyl coating on the wetted surfaces of the fluidicflow path. Typically, the annealing cycle involves subjecting thecoating to 200° C. for 3 hours under vacuum.

The method can also include assessing the polarity of thechromatographic media and selecting the alkylsilyl coating based on thepolarity of the analyte and the chromatographic media. The method canalso include eluting the sample through the fluidic flow path, therebyisolating the analyte.

In some embodiments, the alkylsilyl coating is modified with asilanizing reagent to obtain a desired thickness of the alkylsilylcoating. The silanizing reagent can be a non-volatile zwitterion. Thenon-volatile zwitterion can be sulfobetaine or carboxybetaine. In someembodiments, the silanizing reagent is an acidic or basic silane. Thesilanizing reagent can introduce polyethylene oxide moieties, such asmethoxy-polyethyleneoxy(6-9)silane, the structure of which is shownbelow.

In some aspects, the method of tailoring a fluidic flow path forseparation of a sample including a biomolecule further comprises:pretreating the wetted surfaces of the flow path with a plasma prior todepositing the first coating. In other aspects, the method of tailoringa fluidic flow path for separation of a sample including a metalinteracting analyte further comprises annealing the first coating at atemperature to increase cross-linking in the first coating. In yetanother aspect, the method of tailoring a fluidic flow path forseparation of a sample including a metal interacting analyte furthercomprises annealing the first coating at a temperature to alterhydrophobicity.

In one aspect, the method of tailoring a fluidic flow path forseparation of a sample including a metal interacting analyte furthercomprises performing a second infiltration with a vaporized sourcehaving the Formula II, wherein the features for Formula II are asdescribed above; along and throughout the interior flow path of thefluidic system to form a second coating deposited in direct contact withthe first coating. In one aspect, the step of performing a secondinfiltration in the preceding method further comprises performing anannealing step after depositing the second coating. In another aspect,the preceding method further comprises connecting in fluid communicationwith the flow path at least one coated component selected from the groupconsisting of a sample reservoir container and a frit.

Also provided herein is a method of tailoring a fluidic flow path forseparation of a sample including a metal interacting analyte, the methodcomprising: assessing polarity of the analyte in the sample; selectingan alkylsilyl coating having the Formula I, wherein the features forFormula I are as described above, and desired contact angle based onpolarity assessment; and adjusting the hydrophobicity of wetted surfacesof the flow path by vapor depositing an alkylsilyl having the FormulaIII, wherein the features for Formula III are as described above, andproviding the desired contact angle. In some embodiments of the abovemethod, in addition to assessing polarity of the analyte in the sample,the polarity of a stationary phase disposed within at least a portion ofthe flow path is also assessed and the polarity assessment is obtainedfrom both the polarity of the biomolecule in the sample and thestationary phase.

Methods of Isolating an Analyte

In one aspect, provided herein are methods of isolating an analyte. Themethod includes introducing a sample including a glycan, a peptide, apesticide, or a citric acid cycle metabolite into a fluidic systemincluding a flow path disposed in an interior of the fluidic system. Theflow path includes a first vapor deposited alkylsilyl inert coatinghaving the Formula I described above and a second vapor depositedcoating of the Formula II described above. The sample is eluted throughthe fluidic system, thereby isolating the glycan, peptide, pesticide, orcitric acid cycle metabolite.

The glycan can be a phosphoglycan. The peptide can be a phosphopeptideand the pesticide can be glyphosate. The citric acid cycle metabolitecan be citric acid or malic acid.

When the analyte is a glycan, peptide or pesticide, the alkylsilylcoating of Formula I can be bis(trichlorosilyl)ethane orbis(trimethoxysilyl)ethane and the alkylsilyl coating of Formula II canbe n-decyltrichlorosilane. When the analyte is a citric acid cyclemetabolite, the alkylsilyl coating of Formula I can bebis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane and thealkylsilyl coating of Formula II can be trimethylchlorosilane ortrimethyldimethyaminosilane.

The flow path can be defined at least in part by the interior surface ofa chromatographic system. The flow path can be further defined at leastin part by passageways through a frit of the chromatographic column. Theflow path can be defined at least in part by interior surfaces oftubing. The flow path can be any flow path described herein, forexample, the flow paths described with respect to the chromatographicdevice.

Methods of Improving Baseline Returns

Also provided herein is a method of improving baseline returns in achromatographic system, the method comprising: introducing a sampleincluding an analyte into a fluidic system including a flow pathdisposed in an interior of the fluidic system, the flow path having alength to diameter ratio of at least 20 and comprising a vapor depositedalkylsilyl coating having the Formula I, wherein the features forFormula I are as described above, a thickness of at least 100 angstromsand a contact angle of about 30 degrees to 110 degrees; and eluting thesample through the fluidic system, thereby isolating the biomolecule. Insome embodiments, the method includes a second layer of Formula II orFormula III, wherein the features of Formula II and II are describedabove.

Kits

Also provided here are kits. The kits include a chromatographiccomponents, for example, a chromatographic column, that has been coatedwith an alkylsilyl coating of Formulas I, II, and/or III, as describedabove. Other components can be provided in the kit that can also includethe coatings described herein, for example, the tubing, frits, and/orconnectors. The kit can also include instructions for separatinganalytes, for example, biomolecules, proteins, glycans, peptides,oligonucleotides, pesticides, bisphosphonic acids, anionic metabolites,and zwitterions like amino acids and neurotransmitters.

Exemplary Separations

Separation of Phosphoglygans

The disclosed coatings, which can be vapor deposited, have been found todramatically improve separations of phosphoglycans by hydrophilicinteraction chromatography (HILIC). To demonstrate the significance ofthis, the released N-glycans from a recombinant alpha-galactosidasewhich can be used as an enzyme replacement therapy for Fabry's diseasewere evaluated. This particular type of enzyme is taken up fromcirculation and delivered intercellularly to lysosomes through themannose-6-phosphate pathway, making it important to identify and monitorthe levels of phosphorylated glycans that are present on its surface.Vapor deposition coated stainless steel column tubes along with matchingcoated stainless steel frits were first tested against correspondinguntreated stainless steel hardware. In this instance, two differenttypes of coating chemistries were used. The coating chemistries used tocoat the frits and tubing were VPD #2 and VPD #7. FIG. 4A-4C showfluorescence chromatograms obtained with these types of column hardware.From these data, it was found that use of coated column hardwaresignificantly improved the recovery of each phosphorylated N-glycanspecies. For example, there was a marked increase in the peak area ofMan7-PP, a high mannose glycan with two mannose-6-phosphate residues,mannose-7-bisphosphate. Where Man7-PP could not be detected withstainless steel column hardware, it was easily detected with vapordeposition coated column hardware. This indicated that this species ofN-glycan was interacting with the metallic surfaces of the columnhardware in such a way that prevented it from reaching the detector.When using vapor deposition coated hardware, the peak area ratio ofMan-7-PP to Man5 (a high mannose glycan without phosphorylation) was0.24:1 (FIG. 4A-4C).

The increased recovery of phosphorylated glycans using coated columnhardware and fits shows that adsorption to metallic column hardwaresurfaces is detrimental to recovery. With this in mind, separations werealso performed with vapor deposition coated stainless steel sample flowpath components (FIGS. 5A and 5B). FIG. 6A-6C show fluorescencechromatograms obtained using coated LC system components in conjunctionwith coated stainless steel column hardware. Phosphoglycan recoveryimproved even more with the use of coated column hardware and C₂C₁₀vapor deposition coated flow path components. Most notably, the peakarea ratio of Man7-PP to Man5 increased to 0.8:1 from the ratio of0.24:1 that was obtained by using coated column hardware alone. Theobserved relative abundance for Man7-PP with the coated system andcoated column hardware is indicative of full recovery for thephosphorylated glycans, as can be determined by orthogonal assays toHILIC of RapiFluor-MS labeled released glycans. In sum, these resultsconfirm that the loss of phosphorylated N-glycan species to sample flowpath surfaces can be alleviated with the use of vapor depositioncoatings.

Separation of Other Phosphorylated Molecules

The principles learned from using vapor deposition coatings forphosphoglycan analysis were extended to facilitate the analysis of othertypes of phosphorylated biomolecules. In which case, the coatings havebeen found to be beneficial to improving the recovery of phosphorylatedpeptides under reversed phase chromatography conditions. To demonstratethese recovery advantages, we evaluated a mixture containingphosphopeptides. This particular sample contains three peptides that aresingly phosphorylated and one that is doubly phosphorylated. Vapordeposition coated stainless steel column tubes along with matchingcoated stainless steel frits were first tested against correspondinguntreated stainless steel hardware. FIG. 7A-7C show UV chromatogramsobtained with these types of column hardware. In each case, the additionof the VPD#2, and the VPD#7 coatings increased the recovery of thesingly phosphorylated peptides by at least 13% over the stainless steelalone (FIG. 4A-4C). The impact of coating the chromatographic flow pathwas much more pronounced with the doubly phosphorylated peptide. Whenusing the stainless steel column hardware, there was no detectablerecovery of the doubly phosphorylated peptide. However, when usingeither type of coated column hardware (VPD#2, and the VPD#7), thispeptide became clearly visible in the obtained chromatograms. Thisresult indicates, once again, that vapor deposition coatings can be usedto minimize undesirable interactions with the metallic surfaces ofchromatographic flow paths and in doing so allow for improved analysesof phosphorylated biomolecules.

As such, in one aspect, the vapor deposition coated column hardware isused to improve the recovery of phosphorylated biomolecules duringanalyses by liquid chromatography. In yet another embodiment of thisinvention, vapor deposition coated flow path components are used inconjunction with vapor deposition coated column hardware to improve therecovery of phosphorylated biomolecules during analyses by liquidchromatography.

The effects of this finding have been demonstrated for two examples ofphosphorylated biomolecules, phosphorylated glycans and phosphorylatedpeptides. Phosphorylated biomolecules refers to any molecule naturallyproduced by an organism that contains a phospho group, including but notlimited to phosphorylated proteins and polynucleotides. Furthermore, itis reasonable to envision this disclosure being used to improve liquidchromatographic analyses of smaller biomolecules, including but notlimited to phospholipids, nucleotides and sugar phosphates. Indeed,vapor deposition coated column hardware has been found to be useful inimproving the recovery and peak shape of sugar phosphates andnucleotides. The effects of employing vapor deposition coated versusuntreated column hardware for the reversed phase LC analyses ofglucose-6-phosphate, fructose-6-phosphate, adenosine triphosphate, andadenosine monophosphate are captured in FIGS. 8-11. Interestingly, thesedata indicate that the use of the vapor deposition coated columnhardware can yield a significant improvement in both the overallrecovery and peak shape of these phosphate containing smallbiomolecules. Thus, it is foreseeable that this disclosure could also beused to improve the chromatography of non-biomolecules, such assmall-molecule pharmaceuticals containing either phospho or phosphonatefunctional groups.

Separation of Sialylated Glycans and Molecules Having Carboxylic AcidMoeities

It has additionally been discovered that vapor deposition coatedhardware can be of benefit to mixed mode separations of sialylatedglycans. In such a technique, sialylated glycans can be resolved using astationary phase that exhibits anion exchange and reversed phaseretention mechanisms. It was just recently discovered that a uniqueclass of stationary phase, referred to as charged surface reversed phasechromatographic materials and described in International Application No.PCT/US2017/028856, entitled “CHARGED SURFACE REVERSED PHASECHROMATOGRAPHIC MATERIALS METHOD FOR ANALYSIS OF GLYCANS MODIFIED WITHAMPHIPATHIC, STRONGLY BASED MOIETIES” and published as WO2017/189357(and incorporated herein by reference in its entirety), is ideallysuited to producing these types of separations. The use of a high puritychromatographic material (HPCM) with a chromatographic surface comprisedof a diethylaminopropyl (DEAP) ionizable modifier, a C18 hydrophobicgroup and endcapping on a bridged ethylene hybrid particle has proven tobe an exemplary embodiment for the separation of glycans labeled withamphipathic, strongly basic moieties, like that imparted by the novellabeling reagent described in International Application No.PCT/US2017/028856 (WO2017/189357). This so-called diethylaminopropylhigh purity chromatographic material (DEAP HPCM) stationary phase iseffective in separating acidic glycans as a result of it being modifiedwith a relatively high pKa (˜10) ionizable modifier that yields uniquelypronounced anionic retention.

In an application to DEAP HPCM mixed mode separations of sialylatedglycans, vapor deposition coated hardware has been shown to yieldimproved chromatographic recoveries and peak shapes of glycanscontaining greater than three sialic acid residues. A comparison offluorescence chromatograms for fetuin N-glycans obtained with untreatedstainless steel versus VPD#7 coated hardware is provided in FIG. 12,wherein the effect on peak shape and recovery of tetra- andpenta-sialylated glycans is easily visualized. The observedchromatographic differences are likewise easily quantified. Inparticular, fluorescence peak areas for the most abundant di-, tri-,tetra- and penta-sialylated glycans showed there were indeed verydistinct differences in recoveries (FIG. 13). This testing was also usedto demonstrate that other, chemically unique vapor deposition coatingcould be used with equally good effect. Much like the VPD#7 coatedhardware, VPD#2 and SilcoTek Dursan® coated hardware showed equivalentcapabilities in improving peak shape and recovery of the tetra- andpenta-sialylated N-glycans. Interestingly though, it was not found to benecessary to use a coated flow through needle or column inlet in orderto optimize peak shape and recovery.

As with phosphorylated species, this effect on the chromatography ofsialylated glycans is believed to result from masking the metallicsurface of the hardware and minimizing adsorptive sample losses that canoccur with analytes that exhibit a propensity for metal chelation.However, the origin of the metal chelation is different in that theeffect is a consequence of a glycan carrying multiple carboxylateresidues versus one or two phosphorylated residues. Carboxylatecontaining compounds generally have a weak affinity for metals. Yet,when there are multiple carboxylate moieties present in one molecule, anopportunity for polydentate chelation is created, as is the case withtetra- and penta-sialylated glycans.

Accordingly, in an embodiment of this invention, vapor deposition coatedcolumn hardware is used during liquid chromatography of biomoleculescontaining greater than three carboxylic acid residues as a means toimprove their peak shape and recovery. In yet another embodiment of thisinvention, vapor deposition coated flow path components are used inconjunction with vapor deposition coated column hardware to improve thepeak shape and recovery of biomolecules containing greater than threecarboxylic acid residues.

Separation of Proteins

Certain vapor deposition coatings have also been found to beneficiallyimpact protein reversed phase chromatography. To demonstrate such, weevaluated a paradigmatic protein separation that is very important tothe analysis of biopharmaceuticals, a monoclonal (mAb) subunitseparation with MS-friendly, formic acid modified mobile phase. Usingsuch a test, numerous combinations of column hardware materials havebeen examined. Vapor deposition coated stainless steel column tubesalong with matching coated stainless steel frits were first testedagainst corresponding untreated stainless steel hardware. FIGS. 14A and14B show fluorescence chromatograms obtained with these column hardwarematerials. From these data, it was found that hardware coated withVPD#7, but not hardware coated with VPD#2, was uniquely able to improvethe baseline quality of the model separation, particularly in providingquicker returns to baseline. This improvement to the chromatographicperformance of the separation is underscored by the fact that thechromatogram produced with the VPD#7 coated column also shows higherpeak intensities for some of the subunits. The nature of this baselineissue, as it exists with stainless steel hardware, can be reasoned to bea result of the protein analytes undergoing problematic secondaryinteractions and not homogenously eluting at one particular eluotropicstrength. Interestingly, in this example, the VPD#7 MVD hardware did notappear to significantly improve half height peak capacity nor thecarryover of the columns, which was universally found to be ˜0.9%. Thatis to say, for protein reversed phase chromatography, it would seem thatvapor deposition coatings improve the quality of separationpredominately through affecting baseline properties.

An effect such as this can be very significant to protein reversed phaseseparations, particularly those intended to facilitate detection byonline electrospray ionization (ESI)-mass spectrometry (MS). Often, itis critical to have quick returns to baseline in ESI-MS data given thatit will make the assignment of chromatographic peaks less ambiguous.Signal from previously eluted species will be less abundant andtherefore less confounding in data accumulated for later eluting peaks.With this in mind, 11 additional combinations of column hardwarematerials were screened, using ESI-MS detection as the means toassessing the quality of the data. FIG. 15A presents total ionchromatograms (TICs) for some of these materials, including columnsconstructed with stainless steel alternatives, namely polyether etherketone (PEEK) and a low titanium, nickel cobalt alloy (MP35NLT).Surprisingly, columns constructed of VPD#7 coated hardware were the onlyfound to give uniquely quick returns to baseline. Stainless steel, PEEK,and VPD#2 coated hardware showed comparatively slower returns tobaseline. In addition, control experiments showed that the improvementto baseline quality can be achieved through the use of a VPD#7 coatedfrit alone and that coated tubing is not required to achieve an effect.Further experimentation culminating in the chromatograms of FIG. 15B hasmade it possible to glean additional insights. One of which is that itdoes not matter if the frit has a 0.2 or 0.5 μm porosity or if the VPD#7coating has been thermally cured in the form of an annealing process(resulting in a VPD#8 coating). In contrast, neither a thicker VPD#3coating (˜1800 Å thickness) nor a cured coating (VPD#5) with anincreased contact angle of 90° (up from ˜35°) were able to produce theeffect. Accordingly, VPD#7 coated frits are very unique in their abilitybeing to affect the baseline of the example protein separation. Whilenot limited to theory, it would seem reasonable to suggest that thiseffect derives from the hydrophobicity/contact angle of this coating. Itcould be that these coated frits closely mimic the surface chemistry ofthe reversed phase stationary phase. Consequently, a column with VPD#7coated frits might exhibit adsorption sites (particularly those near thefrit surface) that are more uniform in their chemical properties.Testing has shown that this effect on the protein reversed phaseseparation can be localized to the inlet frit of the column, lendingcredence to this hypothesis (FIG. 16). Indeed, one hydrophobic VPD#7vapor deposition coated frit at the column inlet is sufficient toproduce uniquely quick returns to baseline for the example mAb subunitseparations. Proteins undergo reversed phase chromatography via fairlydiscrete adsorption/desorption events. Consequently, upon loading,protein analytes will be most concentrated at and likewise spend asignificant amount of time at the head of the column, where an interfaceexists between the inlet frit and the packed bed of the stationaryphase. At this interface, a protein analyte would have an opportunity toestablish undesired secondary interactions that would be cumulative toand energetically different than the desired hydrophobic interactionwith the stationary phase. It is plausible that using a frit withsurface properties similar to the stationary phase mitigates anychromatographic problems related to there being energetically andchemically diverse adsorption sites present at this packed bedinterface. While not limited to theory, it may also be possible that afrit, such as the C₂C₁₀ vapor deposition coated inlet frit (e.g., fritcoated with VPD#7), imparts an entirely novel focusing effect to proteinreversed phase separations that cannot be explained by the understandingand descriptions noted above. In addition, it is possible that a frit,such as the VPD#7 vapor deposition coated inlet frit, makes a uniquecontribution to how a stationary phase packs into a column. Use of avapor deposition coated frit as the substrate for building a packedcolumn bed may advantageously impact the properties of a stationaryphase and resultant chromatography.

As such, in an embodiment of this invention, vapor deposition coatedcolumn hardware is used to improve the chromatographic performance ofprotein reversed phase separations. In yet another embodiment of thisinvention, a vapor deposition coating with a contact angle of >90°, morepreferably greater than 100 Å, is used to coat the tubing and frits of acolumn, or chromatographic device, as a means to improve the baselineand/or tailing factors of protein separations.

In a separate embodiment, this invention may utilize a frit materialthat is constructed of a specific polymer, such that an equivalentlyhydrophobic surface is achieved, specifically one with a contact anglegreater than 90°, more preferably greater than 100 Å.Polytetrafluoroethylene (PTFE), polymethylpentene (PMP), high densitypolyethylene (HDPE), low density polyethylene (LDPE) and ultra highmolecular weight polyethylene (UHMWPE) are examples of hydrophobicpolymers that could be suitable for use as the frit or column materialin other embodiments of this invention. In fact, an inlet fritconstructed of porous PTFE (1.5 mm thick, Porex PM0515) was found tofavorably affect protein reversed phase baselines, in a manner similarto that of the previously mentioned VPD#7 vapor deposition coated inletfrit (FIG. 17). Frits of alternative compositions are also relevant tothis invention. In yet another embodiment, parylene, that is polyp-xylene polymer, coatings could be used treat column frits and tothereby improve the properties of a protein reversed phase separation.In addition, glass membranes could be used as the basis of a fritmaterial. Onto the glass membrane substrate, silanes could be bonded toadvantageously manipulate the hydrophobicity and contact angle of thematerial. These and other such membranes could also be used inconjunction with a backing material, like a porous polymer sheet, tolend physical rigidity to the apparatus.

Finally, vapor deposition coated hardware has been found to be ofbenefit to aqueous biomolecule separations, such as protein ion exchangechromatography. When looking to understand the charge heterogeneity of asample, an analyst will often choose to resolve the components of asample by ion exchange. In the case of protein therapeutics, this typeof analysis is performed as a means to interrogate so-called chargevariants, such as deamidiation variants, that can have a detrimentaleffect on the efficacy of the corresponding drug product. Charge variantseparations by way of ion exchange can therefore be critical to theeffectiveness of a characterization approach for a protein therapeutic,most particularly a monoclonal antibody. Being such an importantanalytical approach, protein ion exchange must be robust and able toquickly and reliably yield accurate information.

To this end, ion exchange separations of a monoclonal antibody wereevaluated, and the effects of using uncoated versus vapor depositioncoated column hardware were contrasted. FIGS. 21A-21L presentschromatograms of NIST reference material 8671, an IgG1κ mAb, as obtainedfrom sequential cation exchange separations and repeat injections ofsample. In this evaluation, columns derived from four differentconstructions were tested. These columns varied with respect to bothhardware design and vapor deposition coating. From the observed results,it was most apparent that uncoated hardware showed a prominentconditioning effect, as manifest in there having been low peak areas oninitial injections. While not limited to theory, it is believed that themetallic surfaces of the uncoated column hardware imposed adsorptivelosses on these separations and thereby hindered recovery of the sample.In contrast, vapor deposition coated hardware, both C₂ or C₂-GPTMS-OHchemistries, yielded comparatively high peak areas even on the veryfirst runs of the columns (FIG. 22). That is, coated hardware showed noevidence of requiring a passivation step, giving it the unique advantageof more quickly providing accurate chromatographic data.

Here, it is clear that the noted vapor deposition coatings enhance thechromatographic properties of metallic hardware. Little can be seen inthe way of distinguishing the chromatographic performance of the twotested vapor deposition coatings, namely the C₂ and C₂-GPTMS-OHchemistries. However, the C₂-GPTMS-OH coating has an inordinately lowcontact angle (as does C₂PEO). It is foreseeable that certain types andclasses of biomolecules will require a highly hydrophilic flow path. Onesuch example could indeed be aqueous protein separations in whichhydrophobic interactions could lead to poor recovery or peak tailing. Asa whole, it is believed that vapor deposition coated hardware will showadvantages for numerous forms of aqueous separations, including but notlimited to ion exchange, size exclusion and hydrophobic interactionchromatography, and that the most ideal vapor deposition coating wouldbe one that is very hydrophilic. Accordingly, in an embodiment of thisinvention, a vapor deposition coated column is used to improve therecovery of samples from aqueous chromatographic separations. In a morespecific embodiment, a vapor deposition coating with a contact angleless than 20° is used to improve the recovery of biomolecules in ionexchange, size exclusion or hydrophobic interaction chromatography.

EXAMPLES Example 1

C₂ and C₂C₁₀ Vapor Deposition Coatings

Prior to coating, all metal components are passivated according to anitric acid passivation. Passivated parts and a silicon wafer are thenintroduced to the vapor deposition chamber and vacuum is established.The first step is a 15 minute, 200 Watt, 200 cc/min oxygen plasmacleaning step. Next is the first vapor deposition cycle. Each vapordeposition cycle contains a silane vapor deposition, followed by theintroduction of water vapor for silane hydrolysis. The silane vapor isdelivered at a pressure of 2.0 Torr for 5 seconds, and then the watervapor is delivered at a pressure of 50 Torr for 5 seconds. Followingdelivery, the silane and water is left to react with the substrate for15 minutes. This cycle is repeated to produce the desired number oflayers and coating thickness. An additional processing cycle can beimplemented to functionalize the coating with yet another silane.Moreover, a post coating annealing step can be used to furthercross-link and increase the hydrophobicity of the coating. Typically,the annealing cycle involves subjecting the coating to 200° C. for 3hours under vacuum.

A silicon wafer is used as a coupon to measure the thickness and contactangle of the coating. To measure the thickness, a Gaertner ScientificCorporation stokes ellipsometer model LSE is used. By analyzing thechange in polarization of light, and comparing to a model, the filmthickness can be established. To measure the contact angle, a Ramé-Hartgoniometer model 190 is used. After dropping a controlled amount ofwater onto a perfectly level silicon wafer, optical techniques are usedto measure the contact angle.

Example 2 C₂-GPTMS-OH Vapor Deposition Coatings

Prior to coating, all metal components are passivated according to anitric acid passivation. Passivated parts and a silicon wafer are thenintroduced to the vapor deposition chamber and vacuum is established.The first step is a 15 minute, 200 Watt, 200 cc/min oxygen plasmacleaning step. Next is the first vapor deposition cycle. Each vapordeposition cycle contains a silane vapor deposition, followed by theintroduction of water vapor for silane hydrolysis. The silane vapor isdelivered at a pressure of 2.0 Torr for 5 seconds, and then the watervapor is delivered at a pressure of 50 Torr for 5 seconds. Followingdelivery, the silane and water is left to react with the substrate for15 minutes. This cycle is repeated to produce the desired number oflayers and coating thickness. In this example, thebis(trichlorosilyl)ethane silane is used to build up an adhesion orprimer layer of approximately 800 Å. After C₂ deposition, the3-(glycidoxypropyl)trimethoxysilane is delivered anhydrously to apressure of 0.4 Torr in the vapor deposition chamber. This silane vaporis left to react with the C₂ coated substrate for one hour. This processresults in an epoxide terminated coating, with a contact angle of 50°.After deposition, the next step is to hydrolyze the epoxide groups. Thisis performed either in the liquid phase or the vapor phase, with 0.1Macetic acid. After epoxide hydrolysis, the contact angle is <20°.Contact angle measurements are taken on a silicon wafer using aRamé-Hart goniometer model 190.

Example 3 Alternative Contact Angle Measurement

It is relatively easy to measure the contact angle on the flat siliconwafers using a goniometer. However, not all our substrates have suchsmooth and flat surfaces. Frits can be considered a chromatographycolumn's most important substrate, since the fluidic surface area tomass ratio is higher in the frit than in any other column hardwarecomponent. In order to measure the solid-liquid wetting properties offrit porosity, and confirm the presence of a coating, we can use thebubble point test. The bubble point test is used to determine thelargest pore diameter of a frit structure, and the bubble point pressureis related to this diameter with the following equation:

P=(2γ cos θ)/r

Where,

P=bubble point pressure, Pa (measured)γ=surface tension of test liquid, N/m (known)Θ=contact angle between test liquid and pore material (calculated)r=largest pore radius, m (calculated)

This equation is from ASTM E128 and is derived from the equilibriumcondition of capillary rise.

Using the bubble point test to calculate a contact angle requires twosteps. This first is to test the frit in IPA, and assume a 0 contactangle, since IPA has excellent wetting characteristics. This will yielda maximum pore diameter. The next step is to repeat the experiment withwater as the test liquid, and the known pore radius. This will yield thecontact angle with water, relative to the assumed 0 degree contact angleof IPA. FIG. 18 displays the different bubble point pressures recordedversus coating composition. FIG. 19 displays the derived contact anglesversus coating composition. These values correlate well withmeasurements taken with a goniometer on a flat silicon wafer.

Example 4 Corrosion Performance of Silane Coatings

ASTM G48 Method A is used to rank the relative pitting corrosionperformance of various grades of stainless steel. It consists of placinga part in ˜6% ferric chloride solution for 72 hours, and checking themass loss of your component. The test can be run at room temperature, orat slightly elevated temperatures to increase the corrosion rate. Theferric chloride solution is similar to the environment inside a pitduring “non-accelerated” pitting corrosion; an acidic, oxidizing,chloride containing environment. When an entire part of interest issubmerged in the ferric chloride solution, pitting corrosion is greatlyaccelerated, with normal test times only being 72 hours. FIG. 20displays the corrosion performance of a non-coated column tube, andvarious coatings on a column tube. The improvement ranges from ˜10× to˜100×.

Example 5 HILIC-Fluorescence-MS of Phosphoglycans

A recombinant alpha-galactosidase was diluted to 2 mg/mL. A 7.5 uLaliquot of the protein solution was then added to a 1 mL reaction tubecontaining 15.3 μL of water and 6 μL of buffered 5% RapiGest SFsolution-commercially available from Waters Corporation (Milford, Mass.)(50 mM HEPES-NaOH, pH 7.9). The mixture was placed in a heat block at90° C. for 3 minutes. Thereafter, the reaction tube was allowed to coolat room temperature for 3 minutes. To the reaction tube, 1.2 μL ofPNGase F was then added and incubated at 50° C. for 5 minutes. Afterincubation, the reaction was again allowed to cool at room temperaturefor 3 minutes. To a vial containing 9 mg of RapiFluor-MS reagent, 131 uLof anhydrous DMF was added and vortexed to create a labeling solution. A12 uL volume of this labeling solution was next added to the reactiontube. This labeling reaction was allowed to proceed for 5 minutes toproduce the final sample.

A fully porous amide HILIC stationary phase (1.7 um, 130 Å) was used ina 2.1×50 mm column dimension to chromatograph the samples at a flow rateof 0.4 mL/min and temperature of 60° C. The gradient flow conditionsinitiated with 75.0% organic solvent (Acetonitrile) and 25.0% aqueouseluent (50 mM ammonium formate, pH 4.4) followed by a 11.66 min lineargradient to 54.0% organic/46% aqueous eluent. The column was then cycledthrough an aqueous regeneration step at 100% aqueous mobile phase at aflow rate of 0.2 mL/min for one minute. After the aqueous regeneration,the column was equilibrated at initial conditions for 4 minutes. Specieseluting during the above separations were detected serially viafluorescence (Ex 265/Em 425, 2 Hz) followed by online ESI-MS. Massspectra were acquired with a Xevo G2-XS QToF mass spectrometer operatingwith a capillary voltage of 2.2 kV, source temperature of 120° C.,desolvation temperature of 500° C., and sample cone voltage of 50 V.Mass spectra were acquired at a rate of 2 Hz with a resolution ofapproximately 40,000 over a range of 700-2000 m/z. FIGS. 4A-4C presentcomparisons of this HILIC separation of RapiFluor-MS labeled releasedN-glycans from a recombinant alpha-galactosidase as performed withcolumns constructed of varying coatings and materials. FIGS. 6 Å-6Cpresent comparisons of this HILIC separation as performed with columnsconstructed of varying coatings and materials in conjunction with asample needle and column inlet tubing constructed of varying coatings.

Example 6 RPLC-UV-MS of Phosphopeptides

A vial of phosphopeptide test standard (Waters Corporation, Milford,Mass.) was reconstituted with 50 uL of 0.1% formic acid. A fully porousCSH C18 stationary phase material (1.7 um, 130 Å) was used in 2.1×50 mmcolumn dimensions to chromatograph the samples at a flow rate of 0.2mL/min at a temperature of 60° C. The gradient flow conditions initiatedwith 0.7% organic mobile phase (0.075% formic acid in acetonitrile) and99.3% aqueous mobile phase (0.1% formic acid) followed by a 30 minlinear gradient to 50% organic/50% aqueous. Species eluting during theabove separations were detected serially via UV (220 nm) followed byonline ESI-MS. Mass spectra were acquired with a Xevo G2-XS QToF massspectrometer operating with a capillary voltage of 1.5 kV, sourcetemperature of 100° C., desolvation temperature of 350° C., and samplecone voltage of 50 V. Mass spectra were acquired at a rate of 2 Hz witha resolution of approximately 40,000 over a range of 500-6500 m/z. FIGS.7 Å-7C present comparisons of this reversed phase separation of thephosphopeptide standard performed with columns constructed of varyingcoatings and materials.

Example 7 RPLC/MS of Small Biomolecules (Nucleotides and SugarPhosphates)

RPLC/MS analyses of two nucleotides (adenosine monophosphate andadenosine triphosphate) and two sugar phosphates (glucose-6-phosphateand fructose-6-phosphate) were performed by reversed phase separationswith an organosilica C18 stationary phase according to the methodsparameters noted below. FIGS. 8-11 present comparisons of these reversedphase separations as performed with columns constructed of varyingcoatings and materials.

LC Conditions

-   Columns: BEH C18 130 Å 1.7 μm 2.1×100 mm-   Mobile Phase A: 0.25% Octylamine in H₂O pH adjusted to 9 with acetic    acid-   Mobile Phase B: ACN-   Column Temperature: 35° C.-   Injection Volume: 10 μL (100 ng/mL sample concentrations)-   Sample Diluent: Water-   Detection: Tandem quadrupole mass spectrometer operating in ESI    negative ionization mode and with MRM acquisition.

Gradient Table:

Time (min) Flow Rate (mL/min) % A % B Curve Initial 0.450 95 5 Initial 50.450 75 25 6 10 0.450 75 25 6 20 0.450 50 50 6 21 0.450 5 95 6 22 0.45095 5 6 30 0.450 95 5 6

Example 8 LC-Fluorescence-MS of Highly Sialylated Glycans Using ChargeSurface Reversed Phase Chromatography

RapiFluor-MS labeled N-glycans were prepared from bovine fetuin (SigmaF3004) according to a previously published protocol (Lauber, M. A.; Yu,Y. Q.; Brousmiche, D. W.; Hua, Z.; Koza, S. M.; Magnelli, P.; Guthrie,E.; Taron, C. H.; Fountain, K. J., Rapid Preparation of ReleasedN-Glycans for HILIC Analysis Using a Labeling Reagent that FacilitatesSensitive Fluorescence and ESI-MS Detection. Anal Chem 2015, 87 (10),5401-9). Analyses of these released glycans were performed using aWaters ACQUITY UPLC H-Class Bio LC system and a separation method basedon a previously described charged surface reversed phase chromatographicmaterial described in International Application No. PCT/US2017/028856,entitled “CHARGED SURFACE REVERSED PHASE CHROMATOGRAPHIC MATERIALSMETHOD FOR ANALYSIS OF GLYCANS MODIFIED WITH AMPHIPATHIC, STRONGLY BASEDMOIETIES” (and incorporated by reference). Specifically, RapiFluor-MSlabeled glycans (e.g., glycans labeled with the labeling reagentdiscussed in PCT/US2017/028856 were separated according to a mixed modeseparation using a fully porous (130 Å) 1.7 μm diethylaminopropyl highpurity chromatographic material (DEAP HPCM) in a 2.1×100 mm columnconfiguration. Details of the method are described below. FIGS. 12 and13 present comparisons of this mixed mode separation of sialylatedglycans as performed with columns constructed of varying coatings andmaterials.

LC Conditions

-   Column: DEAP HPCM 130 Å 1.7 μm 2.1×100 mm-   Mobile Phase A: Water and 100 mM Formic Acid/100 mM Ammonium Formate    in 60%-   Mobile Phase B: 100 mM Formic Acid/100 mM Ammonium Formate in 60%    ACN-   Column Temperature: 60° C.-   Injection Volume: 4 μL-   Sample Concentration: 10 pmol/μL-   Sample Diluent: Water-   Fluorescence Detection: Ex 265 nm/Em 425 nm (10 Hz)

Gradient Table:

Time (min) Flow Rate (mL/min) % A % B Curve Initial 0.400 100.0 0.00Initial 24.00 0.400 78.0 22.0 6 24.20 0.400 0.0 100.0 6 24.40 0.400 0.0100.0 6 24.60 0.400 100.0 0.0 6 30.00 0.400 100.0 0.0 6

Example 9

LC/MS of a Reduced, IdeS Digested Monoclonal Antibody (mAb)

Formulated NIST mAb Reference Material 8671 (an IgG1κ) was added to 100units of IdeS and incubated for 30 minutes at 37° C. The resultingIdeS-digested mAb was then denatured and reduced by the addition of 1MTCEP and solid GuHCl. The final buffer composition for thedenaturation/reduction step was approximately 6 M GuHCl, 80 mM TCEP, and10 mM phosphate (pH 7.1). IdeS-digested NIST RM 8671 (1.5 mg/mL) wasincubated in this buffer at 37° C. for 1 hour, prior to being stored at4° C. Reversed phase (RP) separations of the reduced, IdeS-fragmentedmAb were performed to demonstrate the effects of employing differentvapor deposition coated column hardware pieces, namely the column tubeand the frits that enclose the stationary phase into its packing.

A C4 bonded superficially porous stationary phase (2 μm, Rho 0.63, 290Å) was used in a 2.1×50 mm column dimension to chromatograph the samplesat a flow rate of 0.2 mL/min and temperature of 80° C. across a lineargradient consisting of a 20 min linear gradient from 15 to 55% organicmobile phase (aqueous mobile phase: 0.1% (v/v) formic acid in water;organic mobile phase: 0.1% (v/v) formic acid in acetonitrile). Specieseluting during the above separations were detected serially viafluorescence (Ex 280/Em 320, 10 Hz) followed by online ESI-MS. Massspectra were acquired with a Synapt G2-S mass spectrometer operatingwith a capillary voltage of 3.0 kV, source temperature of 150° C.,desolvation temperature of 350° C., and sample cone voltage of 45 V.Mass spectra were acquired at a rate of 2 Hz with a resolution ofapproximately 20,000 over a range of 500-4000 m/z. FIGS. 14-17 presentcomparisons of this reversed phase C4 separation of reduced,IdeS-digested NIST Reference Material 8671 as performed with columnsconstructed of varying coatings and materials.

Example 10 Ion Exchange Chromatography

NIST mAb Reference Material 8671 (an IgG1κ) was separated using columnsconstructed from a 3 μm non-porous cation exchange stationary phasepacked into either uncoated or vapor deposition coated hardware.Separations were performed with an ACQUITY UPLC H-Class Bio instrumentaccording to the experimental conditions outlined below. FIGS. 21 and 22present comparisons of these separations and their resulting data asobtained with columns constructed of varying coatings and materials.

LC Conditions

-   Columns: 3 μm non-porous cation exchange stationary phase in a    2.1×50 mm column dimension-   Sample: NIST mAb Reference Material 8671 diluted to 2.5 mg/mL with    20 mM MES pH 6.0 buffer-   Gradient: 20 mM MES pH 6.0, 10-200 mM NaCl in 7.5 min-   Flow Rate: 0.2 mL/min-   Column Temperature: 30° C.-   Injection Volume: 1 μL (2.1 mm ID columns)-   Detection: 280 nm-   Hardware Design: Hardware A—Identical to ACQUITY UPLC BEH C18 column    hardware    -   Hardware B—A design with an alternative sealing mechanism and        some alternative material compositions.

Example 11 Oligonucleotide Ion Pair RPLC

Testing has shown that flow paths modified with the vapor depositioncoatings of this invention are also helpful in improving oligonucleotideseparations. Example 11 provides evidence of such as observed in theform of improved recoveries and more accurate profiling of a sample'scomposition, particularly with respect to the first chromatogramsobtained with a column.

In this work, a mixture of 15, 20, 25, 30, 35 and 40-mer deoxythymidinewas separated using columns constructed from a 1.7 μm organosilica 130 ÅC18 bonded stationary phase packed into either uncoated or vapordeposition coated hardware. Separations were performed with an ACQUITYUPLC H-Class Bio instrument according to the experimental conditionsoutlined below. FIGS. 23A-F and 24 present comparisons of theseseparations and their resulting data as obtained with columnsconstructed of varying coatings and materials.

LC Conditions

-   Columns: 1.7 μm organosilica 130 Å C₁₈ bonded stationary phase in a    2.1×50 mm column dimension-   Sample: 15, 20, 25, 30, 35 and 40-mer deoxythymidine (0.5 pmol/μL)-   Column Temperature: 60° C.-   Flow Rate: 0.2 mL/min-   Mobile Phase A: 400 mM HFIP, 15 mM TEA in water-   Mobile Phase B: 400 mM HFIP, 15 mM TEA in methanol-   Gradient: 18 to 28% B in 5 min-   Injection volume: 10 μL-   UV Detection: 260 nm

Example 12 RPLC of Citric and Malic Acid

It should also be pointed out that the benefits of this invention arenot limited to only biomolecules or phosphorylated/phospho groupcontaining analytes. In fact, numerous types of so-called “smallmolecules” can be seen to have their separations improved through theadoption of vapor deposition coated flow paths and column hardware. Onenotable class of small molecules corresponds to compounds having acarboxylic acid moiety. By their nature, these are ubiquitous compoundsand some, like citric acid and malic acid, are important metabolites ofliving organisms, given that they are constituents of the Kreb's cycle.

Herein, we have investigated the effects of separating citric acid andmalic acid with untreated versus vapor deposition coated columns. Citricacid and malic acid were analyzed by LC-MS with columns constructed froma 1.8 μm silica 100 Å C₁₈ bonded stationary phase packed into eitheruncoated or C₂C₃ vapor deposition coated hardware. Separations wereperformed with an ACQUITY UPLC I-Class PLUS instrument, and elutinganalytes were detected with a Xevo TQ-S triple quadrupole massspectrometer according to the experimental conditions outlined below.FIGS. 25A-D presents a comparison of these separations and theirresulting data. It can be observed from these results that use of thevapor deposition coated column hardware led to improvements in recoveryand peak shape and thus sizable increases in MS intensity. This isnoteworthy as it highlights the fact that the vapor deposition coatingcan be used to facilitate the development of a more sensitive and moreaccurate quantitation assay of these and other chemically similarcompounds, including but not limited to isocitric acid, α-ketoglutaricacid, succinic acid, fumaric acid, lactic acid, aconitic acid, itaconicacid, oxaloacetic acid, pyruvic acid, pantothenic acid, biotin, andfolic acid. It is reasonable to assume that even zwitterionic smallmolecules would benefit from this invention. This class of compoundsincludes but is not limited to amino acids and neurotransmitters.Likewise, it is envisioned that this invention will be advantageous tobe used to separate and analyze compounds containing metal bindingmoieties, such as cobalamin and the various types of porphyrins. Lastly,these same compounds would exhibit improved separations whether analyzedby RPLC or other modes of chromatography, such as hydrophilicinteraction chromatography (HILIC), ion exchange, or mixed mode LCseparations (i.e. ion exchange/reversed phase or ion exchange/HILIC).

LC Conditions

-   Columns: 1.8 μm silica 100 Å C₁₈ bonded stationary phase in a 2.1×50    mm column dimension-   Sample: Citric acid    -   Malic acid

Column Temperature: 30° C.

-   Flow Rate: 0.5 mL/min-   Mobile Phase A: 0.1% formic acid, water-   Mobile Phase B: 0.1% formic acid, acetonitrile-   Injection volume: 2 μL

Gradient Table:

Time (min) Flow Rate (mL/min) % A % B Curve Initial 0.500 100.0 0.00Initial 0.25 0.500 100.0 0.00 6 2.00 0.500 75.0 25.0 6 2.50 0.500 5.095.0 6 3.00 0.500 5.0 5.0 6 3.10 0.500 100.0 0.0 6

MS Conditions

-   MS1 Resolution: 1 Da-   MS2 Resolution: 0.75 Da-   Capillary Voltage: 1 kV-   Source Offset: 50-   Desolvation Temp.: 600° C.-   Desolvation Gas Flow: 1000 L/hr-   Cone Gas: 150 L/hr-   Nebulizer: 7 bar-   Source Temp.: 150° C.-   MRM (Citric Acid): 191.2>87.1-   MRM (Malic Acid): 133.2>115.2

Example 13 Mixed Mode Chromatography of Pesticides

Glyphosate is non-selective broad spectrum herbicide which is widelyused across the globe as a crop desiccant. Maximum residue limits (MRLs)are enforced globally on various commodities of interest because of thepotential health consequences posed to consumers. Glyphosate and itsmetabolite aminomethylphosphonic acid (AMPA) require unique approachesfor sample preparation and chromatography separation. Various methodscan be employed for quantitation, whether they are based on reversedphase, porous graphitizes carbon, ion chromatography, hydrophilicinteraction chromatography (HILIC) or mixed mode retention mechanisms.No matter the separation mode, assays for glyphosate and other relatedherbicide compounds can prove to be problematic. First, polar pesticidesare difficult to retain on reversed phase columns withoutderivatization. Second, glyphosate interacts with active metal surfaces.As a result, it is notoriously observed in the form of a broad peak orone with pronounced tailing.Herein, we have investigated the separation of glyphosate with untreatedversus vapor deposition coated mixed mode HILIC columns. Glyphosate wasanalyzed by LC-MS with 1.7 μm diethylamine bonded organosilica 130 Åcolumns constructed from either uncoated or C₂C₁₀ vapor depositioncoated stainless steel hardware. Separations were performed with anACQUITY UPLC H-Class Bio coupled with a Xevo TQ-XS triple quadrupolemass spectrometer according to the experimental conditions outlinedbelow.

FIGS. 26A-B and 27A-B show a comparison of coated and uncoated columnperformance for glyphosate in a solvent standard. As seen in FIG. 26B,glyphosate appears as a severely tailing, broad peak. In contrast, asseen in FIG. 26B, glyphosate is separated with much improved peak shapeon the vapor deposition coated column. It can be observed from theseresults that the use of the vapor deposition coated column hardware ledto significant improvements in peak shape, reduced peak widths and thussizable increases in MS intensity (FIGS. 27A-B). It is reasonable toassume that the vapor deposition coated column also yielded higherrecovery. These results are noteworthy as they demonstrate a means todeveloping more sensitive and more accurate quantitation assays forglyphosate and other chemically similar compounds, including but notlimited to pesticides such as Ethephon, 2-Hydroxyethyl phosphonic acid(HEPA), Glufosinate-Ammonium, N-Acetyl-glufosinate,3-Methylphosphinicopropionic acid (MPPA), Aminomethylphosphonic acid(AMPA), N-Acetyl-glyphosate, N-Acetyl-AMPA, Fosetyl-aluminium,Phosphonic acid, Maleic hydrazide, Perchlorate, and Chlorate.

LC Conditions

-   Columns: 1.7 μm diethylamine bonded organosilica 130 Å stationary    phase in a 2.1 x 100 mm column dimension-   Sample: Glyphosate-   Column Temperature: 50° C.-   Flow Rate: 0.5 mL/min-   Mobile Phase A: 0.9% formic acid, water-   Mobile Phase B: 0.9% formic acid, acetonitrile-   Injection volume: 10 μL

Gradient Table:

Time (min) Flow Rate (mL/min) % A % B Curve Initial 0.500 10 90 Initial4.00 0.500 85 15 2 10.0 0.500 85 15 6 16.0 0.500 10 90 1 20.0 0.500 1090 1

MS Conditions

-   MS1 Resolution: 1 Da-   MS2 Resolution: 0.75 Da-   Capillary Voltage: 2.4 kV-   Ionization: ESI−-   Desolvation Temp.: 600° C.-   Desolvation Gas Flow: 1000 L/hr-   Cone Gas: 150 L/hr-   Nebulizer: 7 bar-   Source Temp.: 150° C.-   MRM 1: 168.0>62.6-   MRM 2: 168.0>149.8

Alternatives:

There are a number of alternative methods and uses for the presenttechnology. While the above methods have generally been discussed withrespect to chromatography or the use of a column. Other types of fluidcomponents having an internal flow path may benefit from the presenttechnology. For example, it is generally thought that capillaryelectrophoresis, such as capillary zone electrophoresis, exhibitsrelatively poor reproducibility. Much of the reproducibility issues canbe reasoned to originate from irreproducible surface chemistry on theinner diameter of the tubular capillaries that are used to perform theseparation. A vapor deposition coating on capillaries intended for a CEseparation may therefore circumvent reproducibility issues as it canyield an inordinately thick, rugged coating. The inventions describedherein may consequently be applicable to improving CE separations ofboth small and large biomolecules.

Moreover, while the examples of the described aspects employcomparatively hydrophobic coatings, with water contact angles rangingfrom 15° to 110°, it is reasonable to suggest that some separationscould be enhanced through the application of hydrophilic coatings,including but not restricted to diol, amide/ureido type, andpolyethylene oxide/glycol bondings.

Other analytes, not yet explicitly described, may also benefit fromvapor deposition coated chromatographic flow paths, for instancephosphorothioated oligonucleotides. Nucleic acids inherently containrepeating phosphodiester bonds as part of their backbone. In some case,the phosphodiester backbone is replaced in part with a phosphorothioatebackbone, which can impart in itself unique challenges for a separation.Similarly, intact and proteolytically digested antibody conjugates maybenefit from methods entailing the use of vapor depositionchromatographic flow paths. Lastly, biomolecules containing histidineresidues are likely to benefit from this invention as, likephosphorylated and carboxylate containing residues, they have apropensity for binding to metal.

1. A chromatographic device for separating analytes in a samplecomprising: a sample injector having a sample injection needle forinjecting the sample into the mobile phase; a sample reservoir containerin fluid communication with the sample injector; a chromatography columndownstream of the sample injector, the chromatography column havingfluid connectors; and fluid conduits connecting the sample injector andthe chromatography column; wherein interior surfaces of the fluidconduits, sample injector, sample reservoir container, andchromatography column form a fluidic flow path having wetted surfaces;and wherein at least a portion of the wetted surfaces of the fluidicflow path are coated with a alkylsilyl coating, wherein the alkylsilylcoating is inert to at least one of the analytes in the sample, thealkylsilyl coating having the Formula I:

wherein R¹, R², R³, R⁴, R⁵, and R⁶ are each independently selected from(C₁-C₆)alkoxy, —NH(C₁-C₆)alkyl, —N((C₁-C₆)alkyl)₂, OH, OR^(A), and halo;R^(A) represents a point of attachment to the interior surfaces of thefluidic system; at least one of R¹, R², R³, R⁴, R⁵, and R⁶ is OR^(A);and X is (C₁-C₂₀)alkyl, —O[(CH₂)₂O]₁₋₂₀—,—(C₁-C₁₀)[NH(CO)NH(C₁-C₁₀)]₁₋₂₀-, or —(C₁-C₁₀)[alkylphenyl(C₁-C₁₀)alkyl]₁₋₂₀-.
 2. The chromatographic device of claim1, wherein the alkylsilyl coating has a contact angle of at least 15°.3. The chromatographic device of claim 1, wherein the alkylsilyl coatinghas a contact angle less than or equal to 30° or less than or equal to90°
 4. The chromatographic device of claim 1 further comprising adetector downstream of the chromatography column and wherein the fluidicflow path further comprises the detector.
 5. The chromatographic deviceof claim 4, wherein the detector is a mass spectrometer and the fluidicflow path includes wetted surfaces of an electrospray needle.
 6. Thechromatographic device of claim 1, wherein the fluidic flow path has alength to diameter ratio of at least
 20. 7. The chromatographic deviceof claim 1, wherein the alkylsilyl coating has a thickness of at least100 Å.
 8. The chromatographic device of claim 1, wherein X is(C₂-C₁₀)alkyl.
 9. The chromatographic device of claim 1, wherein X isethyl.
 10. The chromatographic device of claim 1, wherein R¹, R², R³,R⁴, R⁵, and R⁶ are each methoxy or chloro.
 11. The chromatographicdevice of claim 1, wherein the alkylsilyl coating of Formula I isbis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane.
 12. Thechromatographic device of claim 1, further comprising a secondalkylsilyl coating in direct contact with the alkylsilyl coating ofFormula I, the second alkylsilyl coating having the Formula II:

wherein R⁷, R⁸, and R⁹ are each independently selected from—NH(C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkyl, (C₁-C₆)alkenyl, OH, andhalo; R¹⁰ is selected from (C₁-C₆)alkyl, —OR^(B),—[O(C₁-C₃)alkyl]₁₋₁₀O(C₁-C₆)alkyl, —[O(C₁-C₃)alkyl]₁₋₁₀OH and phenyl,wherein said (C₁-C₆)alkyl is optionally substituted with one or morehalo and wherein said phenyl is optionally substituted with one or moregroups selected from (C₁-C₃)alkyl, hydroxyl, fluorine, chlorine,bromine, cyano, —C(O)NH₂, and carboxyl; R^(B) is —(C₁-C₃)alkyloxirane,—(C₁-C₃)alkyl-3,4-epoxycyclohexyl, or —(C₁-C₄)alkylOH; the hashed bondto R¹⁰ represents an optional additional covalent bond between R¹⁰ andthe carbon bridging the silyl group to form an alkene, provided y is not0; and y is an integer from 0 to
 20. 13. The chromatographic device ofclaim 12, wherein y is an integer from 2 to
 9. 14. The chromatographicdevice of claim 12, wherein y is 9, R¹⁰ is methyl, and R⁷, R⁸, and R⁹are each ethoxy or chloro.
 15. The chromatographic device of claim 12,wherein the alkylsilyl coating of Formula II is(3-glycidyloxypropyl)trimethoxysilane, n-decyltrichlorosilane,trimethylchlorosilane, trimethyldimethyaminosilane,methoxy-polyethyleneoxy(1-10) propyl trichlorosilane, ormethoxy-polyethyleneoxy(1-10) propyl trimethoxysilane.
 16. Thechromatographic device of claim 12, wherein the alkylsilyl coating ofFormula II is (3-glycidyloxypropyl)trimethoxysilane followed byhydrolysis.
 17. The chromatographic device of claim 12, wherein thealkylsilyl coating of Formula I and II provides a desired contact angleof about 0° to about 105°.
 18. The chromatographic device of claim 12,wherein the alkylsilyl coating of Formula I is bis(trichlorosilyl)ethaneor bis(trimethoxysilyl)ethane and the alkylsilyl coating of Formula IIis (3-glycidyloxypropyl)trimethoxysilane.
 19. The chromatographic deviceof claim 12, wherein the alkylsilyl coating of Formula I isbis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane and thealkylsilyl coating of Formula II is(3-glycidyloxypropyl)trimethoxysilane followed by hydrolysis.
 20. Thechromatographic device of claim 12, wherein the alkylsilyl coating ofFormula I is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane andthe alkylsilyl coating of Formula II is n-decyltrichlorosilane.
 21. Thechromatographic device of claim 12, wherein the alkylsilyl coating ofFormula I is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane andthe alkylsilyl coating of Formula II is trimethylchlorosilane ortrimethyldimethyaminosilane.
 22. The chromatographic device of claim 12,wherein the alkylsilyl coating of Formula I is bis(trichlorosilyl)ethaneor bis(trimethoxysilyl)ethane and the alkylsilyl coating of Formula IIis methoxy-polyethyleneoxy(3)silane.
 23. The chromatographic device ofclaim 1, further comprising a alkylsilyl coating having the Formula IIIin direct contact with the alkylsilyl coating of Formula I,

wherein R¹¹, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are each independently selectedfrom (C₁-C₆)alkoxy, —NH(C₁-C₆)alkyl, —N((C₁-C₆)alkyl)₂, OH, and halo;and Z is (C₁-C₂₀)alkyl, —O[(CH₂)₂O]₁₋₂₀—, —(C₁-C₁₀)[NH(CO)NH(C₁-C₁₀)] or—(C₁-C₁₀) [alkylphenyl(C₁-C₁₀)alkyl]₁₋₂₀-.
 24. The chromatographicdevice of claim 23, wherein the alkylsilyl coating of Formula III isbis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane.
 25. Thechromatographic device of claim 23, wherein the alkylsilyl coating ofFormula I is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane andthe alkylsilyl coating of Formula III is bis(trichlorosilyl)ethane orbis(trimethoxysilyl)ethane.
 26. The chromatographic device of claim 25,wherein the alkylsilyl coating of I and III has a total thickness ofabout 400 Å. 27-80. (canceled)